Cell populations, methods of transdifferentiation and methods of use thereof

ABSTRACT

The present invention provides methods for the sequential and temporally-regulated administration of pancreatic transcription factors to induce non-pancreatic cells to transdifferentiate and mature along the pancreatic β-cell lineage. The present invention also provides methods for identifying, isolating and enriching transdifferentiation predisposed cells and methods for treating a degenerative pancreatic disorder such as diabetes.

RELATED APPLICATIONS

This application claims priority to and benefit of provisionalapplication U.S. Ser. No. 61/834,759 filed on Jun. 13, 2013 and U.S.Ser. No. 61/834,767 filed on Jun. 13, 2013, the contents of which areherein incorporated by reference in their entireties.

FIELD OF THE INVENTION

The invention generally relates cell populations that are predisposed totransdifferentition and method for to the production of cells having amature pancreatic beta cell phenotype and function.

BACKGROUND OF THE INVENTION

The beta-cells of the islets of Langerhans in the pancreas secreteinsulin in response to factors such as amino acids, glyceraldehyde, freefatty acids, and, most prominently, glucose. The capacity of normalislet beta-cells to sense a rise in blood glucose concentration and torespond to elevated levels of glucose by secreting insulin is criticalto the control of blood glucose levels. Increased insulin secretion inresponse to a glucose load prevents hyperglycemia in normal individualsby stimulating glucose uptake into peripheral tissues, particularlymuscle and adipose tissue.

Individuals in whom islet beta-cells function is impaired suffer fromdiabetes. Insulin-dependent diabetes mellitus, or IDDM (also known asJuvenile-onset or Type I diabetes), represents approximately 10% of allhuman diabetes. IDDM is distinct from non-insulin dependent diabetes(NIDDM) in that only IDDM involves specific destruction of the insulinproducing beta-cells of the islets of Langerhans. The destruction ofbeta-cells in IDDM appears to be a result of specific autoimmune attack,in which the patient's own immune system recognizes and destroys thebeta-cells, but not the surrounding alpha-cells (glucagon producing) ordelta-cells (somatostatin producing) that comprise the islet.

Treatment options for IDDM are centered on self-injection of insulin—aninconvenient and imprecise solution- and thus the development of newtherapeutic strategies is highly desirable. The possibility of islet orpancreas fragment transplantation has been investigated as a means forpermanent insulin replacement (Lacy, 1995; Vajkoczy et al., 1995).Current methodologies use either cadaverous material or porcine isletsas transplant substrates (Korbutt et al., 1997). However, significantproblems to overcome are the low availability of donor tissue, thevariability and low yield of islets obtained via dissociation, and theenzymatic and physical damage that may occur as a result of theisolation process (reviewed by Secchi et al., 1997; Sutherland et al.,1998). In addition are issues of immune rejection and current concernswith xenotransplantation using porcine islets.

It is clear that there remains a critical need to establish alternativesto the treatment of diabetes by self-injection of insulin. While stemcell research has shown promise in this regard, there has not been greatsuccess. There is a need for improved procedures for isolating,culturing, and transdifferentiating non-pancreatic cells to be used inthe treatment of diabetes.

SUMMARY OF THE INVENTION

The present invention provides a method of producing a population ofcells having a mature pancreatic beta cell phenotype and function bycontacting adult mammalian non-pancreatic cells with a pancreatic andduodenal homeobox (PDX-1) polypeptide, or a nucleic acid encoding apancreatic and duodenal homeobox (PDX-1) polypeptide under conditions toallow uptake of the polypeptide, or nucleic acid at a first time period;further contacting the cells with a Pax-4 polypeptide, a NeuroD1polypeptide, or nucleic acid encoding a Pax-4 polypeptide, or nucleicacid encoding a NeuroD1 polypeptide under conditions to allow uptake ofthe polypeptide or nucleic acid at a second time period; and furthercontacting the cells of step with a MafA polypeptide or a nucleic acidencoding a MafA polypeptide under conditions to allow uptake of thenucleic acid at a third time period. The second time period is at least24 hours after the first time period. The third time period is at least24 hours after the second time period. In some embodiments the first,second and third period of time are the same time

Alternatively the invention provides a method of producing a populationof cells having a mature pancreatic beta cell phenotype and function bycontacting adult mammalian non-pancreatic cells with a pancreatic andduodenal homeobox (PDX-1) polypeptide or a nucleic acid encoding apancreatic and duodenal homeobox (PDX-1) polypeptide and a secondpancreatic transcription factor, under conditions to allow uptake of thePDX-1 polypeptide, or nucleic acid and the second pancreatictranscription factor at a first time period; and further contacting thecells with a MafA polypeptide, or a nucleic acid encoding a MafApolypeptide under conditions to allow uptake of the nucleic acid at asecond time period. In some embodiments the second period of time is atleast 2, 3, 4, 5, 6 or 7 days after the first period of time. The secondpancreatic transcription factor is for example, NeuroD1, Pax-4, or Ngn3.

The nucleic acid is a ribonucleic acid or a deoxyribonucleic acid.

Optionally, the cells are further contacted with a nucleic acid encodingSox-9 polypeptide or Sox-9 polypeptide under conditions to allow uptakeof the nucleic acid or polypeptide.

The cells are bone marrow, muscle, spleen, kidney, blood, skin,pancreas, and liver cells. The cells are contacted in vivo. The cellsare contacted in vitro. The population of cells produced by the methodsof the present invention includes at least 0.5 billion cells. In someembodiments, the cells are expanded in culture prior to the contactingwith the polypeptides or nucleic acids.

Also included in the invention are methods of treating a degenerativepancreatic disorder by administering to a subject in need thereof: acomposition comprising a PDX-1 polypeptide or a nucleic acid encoding aPDX-1 polypeptide at a first time period; a composition comprising aPax-4 polypeptide, a NeuroD1 polypeptide, a nucleic acid encoding aPax-4 polypeptide or a nucleic acid encoding a NeuroD1 polypeptide at asecond time period; and a composition comprising MafA polypeptide or anucleic acid encoding a MafA polypeptide at a third time period. Thesecond time period is at least 24 hours after the first time period. Thethird time period is at least 24 hours after the second time period. Insome embodiments the first, second and third period of time are the sametime.

Further provided by the invention are methods of treating a degenerativepancreatic disorder by administering to a subject in need thereof acomposition comprising a PDX-1 polypeptide a nucleic acid encoding aPDX-1 polypeptide and a second pancreatic transcription factor at afirst time period; and a composition comprising a MafA polypeptide or anucleic acid encoding a MafA polypeptide at a second time period. Insome embodiments the second period of time is at least 2, 3, 4, 5, 6 or7 days after the first period of time. The second pancreatictranscription factor is for example, NeuroD1, Pax-4, or Ngn3.

The nucleic acid is a ribonucleic acid or a deoxyribonucleic acid.

Optionally, the subject is further administered a nucleic acid encodingSox-9 polypeptide or Sox-9 polypeptide under conditions to allow uptakeof the nucleic acid or polypeptide.

Also included in the invention are methods of treating a degenerativepancreatic disorder by administering to a subject in need thereof thepopulation of cells produced by the methods of the invention

The degenerative pancreatic disorder is diabetes such as is Type I, TypeII or gestational diabetes. Alternatively, the degenerative pancreaticdisorder is pancreatic cancer or pancreatitis.

The present invention further provides an expression vector including anucleic acid encoding PDX-1 polypeptide and a nucleic acid encoding asecond transcription factor or use in any of the methods for producing apopulation of cells having a mature pancreatic beta cell phenotype ormethods for treating a degenerative pancreatic disorder. The secondtranscription is, for example, NeuroD1, Pax-4, Ngn3, or Sox-9.

Further included in the invention is an enriched population of humancells capable of activating the glutamine synthetase response element(GSRE). At least 5%, 10%, 15%, 20%, 25%, 30% or more of the cells in thepopulation are capable of activating glutamine synthetase responseelement (GSRE). The cells are endothelial cells, epithelial cells,mesenchymal cells, fibroblasts, or liver cells. In some aspects theliver cells are derived from the pericentral liver. Preferably, thecells have active Wnt signaling. At least 5%, 10%, 15%, 20%, 25%, 30% ormore of the cells in the population produce insulin or secrete c-peptidewhen the cells are treated to ectopically express a pancreatictranscription factor, such as Pdx-1, Pax-4, MafA, NeuroD1, or acombination thereof. Optionally, the population of cells express atleast one of Wnt3a; decreased levels of DKK1 or DKK3; decreased levelsof APC; increased activated beta-catenin levels; or STAT3 bindingelements (cis acting factors). In a some aspects the population of livercells isolated from the population of cells the cells express increasedlevels of HOMER1, LAMP3, or BMPR2; or decreased levels of ABCB1, ITGA4,ABCB4, or PRNP.

Also provided by the invention are methods of isolating a population ofcells that have an enriched capacity for transcription factor inducedtransdifferention by providing a heterogeneous population of humancells; introducing a nucleic acid construct comprising a glutaminesynthetase response element (GSRE), or fragment thereof capable ofactivating glutamine synthetase transcription, operatively linked to areporter protein and isolating the cells expressing the reporterprotein. Optionally, the nucleic acid construct further comprises apromoter/enhancer. The reporter protein is a fluorescent protein. Thereporter protein provides resistance to selection pressure. The cellsare endothelial cells, fibroblasts, mesenchymal or liver cells. Theliver cells are derived from the pericentral liver.

Optionally, the method further comprises culturing the isolated cells.

Also included in the invention is the population of cells isolated bythe methods of a according to the invention.

In other aspects the invention includes a method of treating oralleviating a symptom of a pancreatic disorder by introducing apancreatic transcription factor to the cell population isolatedaccording to the methods of the invention administering the cellpopulation to a subject in need thereof. The pancreatic disorder isdiabetes or pancreatitis. The pancreatic transcription factor is Pdx-1,Pax-4, MafA, NeuroD1, or a combination thereof.

Further included in the invention are method of producing a populationof cells having a mature pancreatic beta cell phenotype and function bycontacting the population of cells isolated according to the inventionwith a pancreatic and duodenal homeobox (PDX-1) polypeptide, or anucleic acid encoding a pancreatic and duodenal homeobox (PDX-1)polypeptide under conditions to allow uptake of the polypeptide, ornucleic acid at a first time period; further contacting the cells with aPax-4 polypeptide, a NeuroD1 polypeptide, or nucleic acid encoding aPax-4 polypeptide, or nucleic acid encoding a NeuroD1 polypeptide underconditions to allow uptake of the polypeptide or nucleic acid at asecond time period; and further contacting the cells of step with a MafApolypeptide or a nucleic acid encoding a MafA polypeptide underconditions to allow uptake of the nucleic acid at a third time period.The second time period is at least 24 hours after the first time period.The third time period is at least 24 hours after the second time period.In some embodiments the first, second and third period of time are thesame time

Alternatively the invention provides a method of producing a populationof cells having a mature pancreatic beta cell phenotype and function bycontacting the population of cells isolated according to the inventionwith a pancreatic and duodenal homeobox (PDX-1) polypeptide or a nucleicacid encoding a pancreatic and duodenal homeobox (PDX-1) polypeptide anda second pancreatic transcription factor, under conditions to allowuptake of the PDX-1 polypeptide, or nucleic acid and the secondpancreatic transcription factor at a first time period; and furthercontacting the cells with a MafA polypeptide, or a nucleic acid encodinga MafA polypeptide under conditions to allow uptake of the nucleic acidat a second time period. In some embodiments the second period of timeis at least 2, 3, 4, 5, 6 or 7 days after the first period of time. Thesecond pancreatic transcription factor is for example, NeuroD1, Pax-4,or Ngn3.

The nucleic acid is a ribonucleic acid or a deoxyribonucleic acid.

Optionally, the cells are further contacted with a nucleic acid encodingSox-9 polypeptide or Sox-9 polypeptide under conditions to allow uptakeof the nucleic acid or polypeptide.

The invention provides a nucleic acid construct comprising one or moreglutamine synthetase response elements (GSRE), operably linked to apromoter and a reporter protein. The promoter is a weak promoter. Thenucleic acid construct further contains a transcription factor. Thetranscription factor is a pancreatic transcription factor such as,Pdx-1, Pax-4, MafA, or NeuroD1. Also included in the invention is avector contacting the nucleic acid construct of the invention. Thevector is an adenoviral vector.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, suitable methods andmaterials are described below. All publications, patent applications,patents, and other references mentioned herein are incorporated byreference in their entirety. In case of conflict, the presentspecification, including definitions, will control. In addition, thematerials, methods, and examples are illustrative only and not intendedto be limiting.

Other features and advantages of the invention will be apparent from thefollowing detailed description, and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows that Pdx-1 expression in human liver cells in vitro inducesgradual activation of pancreatic hormone expression. (A) Insulin (INS);(B) glucagon (GCG); (C) somatostatin (SST); and (D) otherpancreas-specific transcription factors (NKX6.1, ISL1, PAX4, MAFA,NeuroD1, NeuroG3). The results were normalized to β-actin geneexpression within the same cDNA sample and are presented as the mean±SEof the relative expression versus control virus treated cells on thesame day. n≧4 in two independent experiments (*p<0.05, **p<0.01).

FIG. 2 shows that ectopic co-expression of pancreatic transcriptionfactors (pTFs) Pdx-1, Pax4, and MafA in human liver cells in vitropromotes (pro)insulin secretion, compared to that induced by each of thepTFs alone. (A) Immunofluorescence (IF) staining shows expression ofpTFs: Pdx-1 (left panel), Pax4 (middle left panel), MafA (middle rightpanel) and a merge of the 3 pTFs (right panel), with arrows indicatingcells expressing all three pTFs. (B) Luciferase assay insulin promoteractivation by the indicated pTFs; β-gal was used as a control. Resultsare expressed as Relative Light Unit (RLU)/mg protein. Each data pointrepresents the mean±SE of at least two independent experiments, *p<0.05,**p<0.01 in comparison to control virus treated cells, (n>4). (C)Immunofluorescence staining shows insulin-positive cells after ectopicexpression of the indicated pTFs. Original magnification ×20.Quantification of IF staining in table (right). The percent ofinsulin-positive cells was calculated by counting at least 500 positivecells from at least two independent experiments. (D) Insulin secretionafter incubation with the indicated concentrations of glucose wasdetected by radioimmunoassay. *p<0.05, n≧12 in five independentexperiments. The significance represents the differences between tripleinfection and all other treatments.

FIG. 3 shows the effects of concerted and sequential expression of pTFsPdx-1, Pax4, and MafA on pancreatic β-cell maturation. (A) A schematicdemonstrating the order of infection of pTFs (treatments B-E) or controlvirus (Ad-CMV-β-gal, treatment A). (B) Immunofluorescence staining forinsulin: treatment B (left panel), treatment C (middle panel), treatmentD (right panel). Original magnification is at ×20. Quantification ofstaining (percent) is indicated below each image. The percent of insulinpositive cells were calculated by counting at least 1000 positive cellsfrom at least two independent experiments. (C) Insulin and (D) C-peptidesecretion after incubation with the indicated concentration of glucosewas measured by radioimmunoassay. Infection treatments are indicated onthe X-axis and explained in Table 3A. *p<0.05, **p<0.01, compared tocontrol virus treated cells; n≧12 in 5 independent experiments. (E)Expression levels of the indicated endogenous pancreas-specifictranscription factors after the indicated treatments (X-axis) weremeasured by RT-PCR. CT values are normalized to β-actin gene expressionwithin the same cDNA sample. Results are presented as relative levels ofthe mean±SE of the relative expression versus control virus treatedcells, *p<0.05 n≧8 in 4 independent experiments. The arrow points thespecific decrease in Isl-1 expression level under treatment C.

FIG. 4 shows three graphs demonstrating transdifferentiation efficiency,indicating hierarchical sequential order of infection (treatment C) ismost efficient. (A) Insulin promoter activation was measured byluciferase assay after the indicated infection treatments. Results areexpressed as Relative Light Unit (RLU)/mg protein. Each data pointrepresents the mean±SE of at least two independent experiments, *P<0.05,**P<0.01, compared to control virus treated cells, (n>4). (B) Analysisof glucose transporter 2 (GLUT2) expression levels by RT-PCR wasperformed after the indicated infection treatments. CT values arenormalized to β-actin gene expression within the same cDNA sample.Results are presented as relative levels of the mean±SE compared tocontrol virus treated cells. *P<0.05, compared to control virus treatedcells n≧8 in 4 independent experiments. (C) Expression levels ofprohormone convertase 2 (PC2; PCSK2) were determined by RT-PCR after theindicated infection treatments. CT values are normalized to β-actin geneexpression within the same cDNA sample. Results are presented asrelative levels of the mean±SE compared to control virus treated cells**P<0.01, n≧8 in 4 independent experiments.

FIG. 5 shows two graphs demonstrating c-peptide secretion afterhierarchical sequential order of infection (treatment C). (A) C-peptidesecretion was measured by radioimmunoassay static incubation for 15 minat 0, 5, 10, 15, 20 mM glucose in cells treated by the direct“hierarchical” sequential order (treatment C) *P<0.05, n≧7 in 3independent experiments. (B) C-peptide secretion was measured byradioimmunoassay over 13 or 28 days in serum free media supplementedwith insulin, transferrin and selenium (ITS), before being analyzed forc-peptide secretion. *P<0.05, **P<0.01, n≧5 in 2 independentexperiments. The significance represents the differences compared to thestandard protocol (treatment C on day 6).

FIG. 6 is four graphs showing the individual role of the pTFs in thetransdifferentiation process, using treatment C infection order andexclusion of each pTF (C-Pdx1, exclusion of Pdx1; C-Pax4, exclusion ofPax4; and C-Mafa, exclusion of Mafa). (A) Insulin promoter activationwas measured by luciferase assay. Results are presented mean±SE, *p<0.1,**p<0.05 compared to the direct “hierarchical” sequential infectionorder (treatment C), n≧6 in three independent experiments. (B) C-peptidesecretion after incubation for 15 minutes with the indicatedconcentrations of glucose and measured by radioimmunoassay. *=p<0.05,**=p<0.01 in compared to the direct “hierarchical” sequential infectionorder (C), n≧6 in three independent experiments. (C) Expression levelsof pancreatic enzymes were measured by RT-PCR: glucose transporter 2(GLUT2); glucokinase (GCK); and prohormone convertase (PCSK2). (D)Expression levels of the indicated endogenous pancreatic transcriptionfactors were measured by RT-PCR. CT values are normalized to β-actingene expression within the same cDNA sample. Results are presented asrelative levels of the mean±SE compared to “hierarchy sequentialinfection” treated liver cells. *p<0.05, **p<0.01, n≧6 in threeindependent experiments.

FIG. 7 shows three graphs showing the effects of Isl1 expression on3-cell maturation of transdifferentiated liver cells after infection by“hierarchical” sequential order (treatment C). (A) Expression levels ofinsulin were measured by RT-PCR. CT values are normalized to β-actingene expression within the same cDNA sample. Results are presented asrelative levels of the mean±SE compared to control virus treated cells.*P<0.05, n≧6 in 3 independent experiments. (B) Insulin secretion wasmeasured by radioimmunoassay. **P<0.01, n≧6 and compared to the direct“hierarchical” sequential infection order (C), n≧6 in 3 independentexperiments. (C) Expression level of glucose transporter 2 (Glut2) wasmeasured by RT-PCR.

FIG. 8 shows the individual role of pTFs in promoting thedifferentiation of cells to produce glucagon (α) and somatostatin (δ)using hierarchical order of infection (treatment C) and exclusion ofeach pTF. Expression levels of pancreatic hormones glucagon (GCG) (A andB) and somatostatin (SST) (A and D) was determined by RT-PCR after theindicated infection treatments. (C) Expression levels of cell-specifictranscription factors ARX and BRAIN4 were also measured by RT-PCR forthe indicated infection treatments. (E) Expression levels ofsomatostatin (SST) were determined by RT-PCR after additional infectionwith Isl1 (100 MOI). CT values (for A, B, C, and D) are normalized toβ-actin gene expression within the same cDNA sample. Results arepresented as relative levels of the mean±SE compared to control virustreated cells (a) or to “hierarchy sequential infection” treated livercells (b-e). *P<0.05, **P<0.1, n≧6 in 3 independent experiments. (F)Immunofluorescence staining for somatostatin after treatment C infection(left panel), and after treatment C infection with additional Isl1infection (right panel). Original magnification×20. (G)Immunofluorescence staining for somatostatin and insulin showing thatthe sequential administration of transcription factors in a directhierarchical manner results in increased maturation of thetransdifferentiated cells along the beta-like-pancreatic lineage

FIG. 9 shows a schematic representation of the proposed mechanism ofpancreatic transcription factor-induced transdifferentiation from liverto pancreas. The concerted expression of the three pTFs results inincreased number of transdifferentiated liver cells compared to each ofthe factor's individual effect (B). The sequential administration oftranscription factors in a direct hierarchical manner results inincreased maturation of the Transdifferentiated cells along thebeta-like-pancreatic lineage

FIG. 10. Pdx-1-induced IPCs' activation in mice in vivo is restricted tocells adjacent to the central veins which are characterized by GSexpression.

Immunohistochemical analysis of Pdx-1 (A) and insulin (B) 14 days afterAd-CMV-PDX-1 administration. Arrows indicate positive cells, mostlylocated at the proximity of central veins (cv). (C&D) analysis of GSexpression in human (C) and mice (D) livers indicating the expression ofGS at the 1-2 cell layers adjacent to the central veins. Originalmagnification ×.400

FIG. 11. GSRE contains Wnt signaling responding element-TCF-LEF bindingsite. A schematic presentation of GSRE indicating the presence ofTCF-LEF and STAT 5 binding sites.

FIG. 12. The GSRE targets subpopulation of human liver cells in vitro.(A&D) Schematic presentations of Ad-GSRE-TK-Pdx-1 or GFP recombinantadenoviruses. Liver cells were infected with Ad-GSRE-TK-Pdx-1 (C) orwith Ad-CMV-Pdx-1 (B). Immunoflorescent analysis of Pdx-1 expressionindicated that 13±2% of the human liver cells infected byAd-GSRETK-Pdx-1 (C) while 70±12% of Ad-CMV-Pdx-1-treated cells (B)expressed the ectopic nuclear factor (rabbit anti-Pdx-1, generous giftfrom C. Wright, pink; B&C, respectively). Similar results were obtainedusing Ad-GSRE-TK-eGFP; ˜15% of the cells were positive to eGFP (E&F).Ad-CMV-eGFP infection resulted in about 75-80% eGFP positive cellswithin 3-4 days (data not presented)

FIG. 13. The GSRE targets transdifferentiation-prone cells. Liver cellswere infected with Ad-GSRE-TK-Pdx-1 (B) or with Ad-CMV-Pdx-1 (A) for 5days. (A&B), Immunoflorescent analysis of co-staining of insulin (Guineapig anti-insulin, Dako, green) and (Pdx-1 rabbit anti-Pdx-1, generousgift from C. Wright, pink). (C) Statistical analysis o activation ofinsulin in the treated cells; Ad-GSRE-TK-Pdx-1 activated insulinproduction in 50%, whereas Ad-CMV-Pdx-1 only in 5% of the Pdx-1-positivecells. Blue—DAPI, nuclear staining; original magnification×20.

FIG. 14. In vitro lineage tracing for GSRE activating human cells. (A) Aschematic presentation of the lentivirus vectors. (B) Adult human livercells at passages 3-10 were infected with the dual lentivirus system.Liver cells were imaged 10 days after infection for DsRed2 (red) or eGFP(green) fluorescence. (C) The cells were sorted by afluorescence-activated cell sorter (FACS; Aria cell sorter; BectonDickinson, San Jose, Calif.) with a fluorescein isothiocyanate filter(530/30 nm) for eGFP and a Pe-Texas Red filter (610/20 nm) for DsRed2.(D&E). The separated cells were cultured separately for several passages(original magnification×10).

FIG. 15. eGFP+ and DsRed2+ cells efficiently proliferate in vitro with asimilar rate of proliferation and similar infection capacity. Theseparate populations of cells were cultured separately for ˜1 month. Theproliferation rate of each group was analyzed (A). eGFP+ (B&C) andDsRed2+ (D&E) cells were infected with Ad-CMV-β-gal (B&D) or withAd-CMV-Pdx-1 (C&E) for 3 days Immunofluorescent analysis usinganti-Pdx-1 (blue) indicated that almost 80% of both eGFP and DsRed2cells were infected by the adenovirus.

FIG. 16. eGFP+ cells respond more efficiently than DsRed2+ cells topTFs-induced transdifferentiation. The two groups were similarly treatedwith soluble factors and pTFs: Ad-Pdx-1+Ad-Pax-4+ad-MafA or a controlvirus (Ad-β-gal) for 6 days. β-cell-like characteristics and functionwas compared in the separated groups: (A) at the molecular level,insulin and glucagon gene expression was studied by Quantitativereal-time compared to the control-treated cells. Cultured pancreatichuman islet cells (Passage 3) used as a positive control. (B&C) At thefunctional level, glucose-regulated insulin secretion was analyzed bystatic incubations at low followed by high glucose concentrations (2 mMand 17.5 mM glucose in KRB, respectively). Insulin (B) and C-peptide (C)secretion were measured using the human insulin radioimmunoassay kit(DPC; n≧8 from 3 different experiments) or human c-peptideradioimmunoassay kit (Linco n≧8 from 3 different experiments. *P<0.01compared to the DsRed2+ cells, using Student's t-test analysis.

FIG. 17. Higher transdifferentiation efficiency in eGFP+ population isstable with increasing passages in culture. The two groups proliferatedseparately after sorting and were similarly treated with pTFs(Ad-Pdx-1+Ad-Pax-4+ad-MafA and soluble factors) after a few passages(5-7 passages post sorting) or a higher number of passages (10-12passages post sorting). Regulated insulin secretion was analyzed bystatic incubations at low followed by high glucose concentrations (2 mMand 17.5 mM glucose in KRB, respectively). Insulin secretion is measuredusing the human insulin radioimmunoassay kit (DPC; n≧6 from 2 differentexperiments). No statistical significant differences were detectedbetween the low and high number of passages in both population of cells,suggesting a persistent tendency of eGFP tagged cells to undergo pTFsinduced transdifferentiation along the β-cell lineage and function.

FIG. 18. Differential gene expression profiles of eGFP+ and DsRed2+cells performed by microarray analyses and analyzed according to DAVIDBioinformatics Resources 6.7 Four Percent of the differential genesbelong to the Wnt signaling pathway.

FIG. 19. The active Wnt signaling promotes liver to pancreastransdifferentiation. Adult human liver cells were treated withAd-CMV-Pdx-1 and soluble factors, as previously reported, supplementedwith Wnt3A (50 ng/ml R&D or DKK3 (3 μg/ml R&D). After 5 days, insulinsecretion was analyzed by static incubations at low followed by highglucose concentrations (2 mM and 17.5 mM glucose in KRB, respectively).Insulin secretion is measured using the human insulin radioimmunoassaykit (DPC; n≧8 from 3 different experiments) and compared to untreatedcells (Cont). *p<0.01 compared to Ad-CMV-Pdx-1 alone, using Student'st-test analysis.

FIG. 20. Blocking Wnt signaling pathway abolishes thetransdifferentiation of eGFP+ cells. eGFP cells were Ad-CMV-Pdx-1 or acontrol virus (Ad-CMV-β-gal) for 5 days supplemented with DKK3 (0.5μg/ml R&D). Pancreatic hormones gene expression was studied byQuantitative real-time RT-PCR compared to the control-treated cells.

FIG. 21. eGFP+ cells express lower levels of APC and higher levels ofactive β-catenin than DsRed2+ cells. (A) APC and DKK1 expression ismarkedly increased in DsRed2+ cells. This may further suggest that thesecells express higher levels of Wnt signaling pathway repressors comparedwith the eGFP+ cells. n≧6 from 2 different experiments *p<0.01 inDsRed2+ compared to eGFP+ cells, using Student's t-test analysis. (B)Western blot analysis using a specific antibody for activated β-catenin(anti-ABC clone 8E7, Millipore, 1:2000) in eGFP and DsRed2 positive cellextracts. β-actin (SC-1616, Santa Cruz, 1:1000) was used as anormalizing protein. (C) Quantification of the β-catenin protein levelswas performed using ImageJ 1.29x software.

DETAILED DESCRIPTION OF THE INVENTION

Transcription factors (TFs) have been shown to inducetransdifferentiation in numerous cell lineages. As referred to herein,“transdifferentiation” refers to the process by which a first cell typeloses identifying characteristics and changes its phenotype to that of asecond cell type. In some embodiments, the first and second cells arefrom different tissues or cell lineages. Preferably,transdifferentiation involves converting a mature or differentiated cellto a different mature or differentiated cell. Specifically,lineage-specific transcription factors (TFs) have been suggested todisplay instructive roles in converting adult cells to endocrinepancreatic cells (Meivar-Levy et al, 2006; Meivar-Levy et al, 2010;Yechoor et al, 2010; Russ et al, 2011), neurons (Vierbuchen et al, 2010;Ambasudhan et al, 2011; Pang et al, 2011), hematopoietic cells (Szabo etal, 2010) and cardiomyocyte lineages (Ieda et al, 2010), suggesting thattransdifferentiation processes occur in a wide spectrum of milieus. Inall transdifferentiation protocols, the ectopic TFs serve as a shortterm trigger to a potential wide, functional and irreversibledevelopmental process (Ber et al, 2003; Meivar-Levy et al, 2003;Meivar-Levy et al, 2006). Numerous studies suggested that ectopicexpression of individual TFs activate a desired alternate repertoire andfunction, in a process involved with the activation of additionalrelevant otherwise silent TFs. However, the time course, the relativelevels and the hierarchy, or order, of the induced TFs, remains unknown.

By exploiting the relative insufficiency of the endogenous transcriptionfactor (TFs) induction by introducing individual ectopic TFs, thepresent invention relates transdifferentiation as a sequential andtemporally controlled process which is affected by a hierarchicalnetwork of TFs.

The present invention is based on the finding that TF-induced liver topancreas transdifferentiation is a gradual and consecutive process.Importantly, only sequential administration of pancreatic TFs but nottheir concerted expression selectively drives lineage specificationprograms within the endocrine pancreas. Sequential expression ofpancreatic TFs in a direct hierarchical mode has been shown to beobligatory for transdifferentiated cell maturation along the β-celllineage. Specifically, a role for the pancreatic β-cell specifictranscription factor MafA has been identified in the final stage of thetransdifferentiation process. At this stage, MafA promotes thematuration of transdifferentiated liver cells along the β-cell lineage,in a process associated with Isl1 and somatostatin repression.

The findings described herein suggest fundamental temporalcharacteristics of transcription factor-mediated transdifferentiationwhich could contribute to increasing the therapeutic merit of usingTF-induced adult cell reprogramming for treating degenerative diseasesincluding diabetes.

Pancreatic transcription factor (pTFs), such as Pdx-1, NeuroD1, Ngn-3and Pax4, activate liver to pancreas transdifferentiation andindividually induce amelioration of hyperglycemia in diabetic mice(Ferber et al, 2000; Ber et al, 2003; Kojima et al, 2003; Koizumi et al,2004; Kaneto et al, 2005; Kaneto et al, 2005). Moreover, using an invitro experimental system of adult human liver cells, we previouslydemonstrated that Pdx-1 activates the expression of numerous β-cellspecific markers and induces glucose regulated secretion of processedinsulin (Sapir et al, 2005; Meivar-Levy et al, 2007; Aviv et al, 2009;Gefen-Halevi et al, 2010; Meivar-Levy et al, 2011). The induced processwas associated with the expression of numerous key endogenous pTFs andamelioration of hyperglycemia was demonstrated upon transplantation ofthe transdifferentiated adult human liver cells in diabetic mice (Sapiret al, 2005). However, numerous other studies have indicated that usingcombinations of several key TFs markedly increases the reprogrammingefficiency compared to that induced by the ectopic expression ofindividual TFs (Kaneto et al, 2005; Tang et al, 2006; Song et al, 2007;Wang et al, 2007; Gefen-Halevi et al, 2010 Zhou et al, 2008; Vierbuchenet al, 2010; Ambasudhan et al, 2011; Pang et al, 2011). This suggests apotential restricted capacity of the individual ectopic factors toactivate the endogenous complementing TFs to sufficient levels neededfor an efficient transdifferentiation process (Kaneto et al, 2005; Zhouet al, 2008; Ambasudhan et al, 2011; Pang et al, 2011). Targeteddisruption or temporal mis-expression of pancreatic transcriptionfactors during pancreas organogenesis hampers pancreas development aswell as islet cells differentiation and function (Nishimura et al,2009). By exploiting the relative insufficiency of the endogenous TFsinduction by individual ectopic TFs, the present invention is related totransdifferentiation as a sequential and temporally controlled processwhich is affected by a hierarchical network of TFs.

Pancreatic specification is initiated by the homeobox transcriptionfactor Pdx1, which is also required for β-cell function in adults(Offield et al, 1996; Stoffers et al, 1997). The endocrinedifferentiation is then mediated by the basic helix-loop-helix factorNgn3 (Gradwohl et al, 2000). The paired homeobox factors Pax4 and Arx,have been implicated as key factors in the segregation of the differentendocrine cell types (Collombat et al, 2003; Brun et al, 2008). Thefinal maturation along the β-cell lineage and function is attributed toselective expression of MafA in β-cells in the adult pancreas (Kataokaet al, 2002).

The present invention is based in part on the surprising finding thathuman liver cells can be directly transdifferentiated to produce anentirely different cell type, pancreatic hormones producing cellsincluding beta-cells. Application of select transcription factors in atemporally-regulated sequence induced the transdifferentiation of adultliver cells to functional mature beta-cells. The invention solves theproblem of producing large populations of insulin-producing cells, orpancreatic beta-cells, by providing methods for expanding andtransdifferentiating adult cells. The compositions comprising the selecttranscription factors or the generated population of transdifferentiatedpancreatic cells can be used for treating a pancreatic disorder usingthe methods described herein.

Previous efforts to transdifferentiate non-pancreatic cells topancreatic cells, such as beta-cells, utilize either only onetranscription factor or the concerted or simultaneous administration ofmore than one pancreatic transcription factor. The invention describedherein provides methods for an ordered, sequential administration ofspecific transcription factors at defined timepoints. Furthermore, themethods described herein substantially increase the transdifferentiationefficiency compared to that induced by each of the individualtranscription factors alone.

The present invention further provides a population of cells whichpossess increased transdifferentiation capacity. These cells arecharacterized by (1) potential cell membrane markers, (2) possessing thecapacity to activate glutamine synthetase regulatory element (GSRE), and(3) by being uniquely equipped with active Wnt-signaling. At least 30%of the cells in the population are capable of activating GSRE. Forexample the cells are endothelial cells, epithelial cells, mesenchymalcells, fibroblasts, or liver cells. Preferably, the cells are humancells. In some embodiments, the cells can be transdifferentiated alongthe pancreatic lineage to mature pancreatic cells with pancreaticfunction. In other embodiments, the cells can be transdifferentiatedalong the neural lineage to neural cells.

Thus, the present invention also solves the problem of previoustransdifferentiation or reprogramming protocols that often haverestricted efficiency. For example, although ectopic expression of keypancreatic transcription factors results in expression in each hostcell, only up to 15% of the cells are successfully transdifferentiatedto exhibit pancreatic function.

The present invention also provides methods for isolating the populationof cells with enriched or increased transdifferentiation capacity. Forexample, one method for isolating these cells is by sorting out cellswhich activate GFP expression operatively linked to the glutaminesynthetase regulatory element, or a fragment thereof, thereby isolatingthose cells that can activate GSRE. The cells may be sorted by FACS andcan be propagated in culture, separately from the rest of the cells, forrapid expansion of the cells with enriched transdifferentiationcapacity. The population of cells with enriched capacity fortransdifferentiation is only a small proportion of the cells that makeup the tissue in vivo. For example, in a given tissue or population ofcells, the population of cells with enriched capacity fortransdifferentiation is only about less than 1%, 2%, 3%, 4%, 5%, about10%, about 15%, of the entire population of cells in a given tissue.Therefore, the present invention also provides methods for the isolationof said cells with increased transdifferentiation capacity from cellsthat do not have increased transdifferentiation capacity. Accordingly,the present invention provides the advantage of a cell population with agreater proportion of cells that have increased transdifferentiationcapacity to increase the efficiency of transdifferentiation to providetransdifferentiated cells for treatment of various diseases ordisorders.

It will be obvious to those skilled in the art that various changes andmodifications may be made to the methods described herein within thespirit and scope of the invention.

Methods of Producing Pancreatic Beta-Cells

The present invention provides methods for producing cells that exhibita mature pancreatic beta cell phenotype by contacting adult mammaliannon-pancreatic cells with pancreatic transcription factors, such asPDX-1, Pax-4, NeuroD1, and MafA, at specific timepoints. In someembodiments, the methods comprise contacting an adult mammaliannon-pancreatic cell with PDX-1 at a first time period; contacting thecells from the first step with Pax-4 at a second time period; andcontacting the cells from the second step with MafA at a third timeperiod. In one embodiment, the methods comprise contacting an adultmammalian non-pancreatic cell with PDX-1 at a first time period;contacting the cells from the first step with NeuroD1 at a second timeperiod; and contacting the cells from the second step with MafA at athird time period. In another embodiment, the methods comprisecontacting an adult mammalian non-pancreatic cell with PDX-1 and asecond transcription factor at a first time period and contacting thecells from the first step with MafA at a second time period. Thetranscription factors may be polypeptides, ribonucleic acids or nucleicacids encoding the transcription factor polypeptides. For example, thetranscription factors provided together with PDX-1 are Pax-4, NeuroD1,Ngn3, or Sox-9. Preferably, the transcription factor is NeuroD1.

In one aspect, the methods described herein further comprise contactingthe cells at, before, or after any of the above steps with thetranscription factor Sox-9.

In one aspect, the second time period is at least 24 hours after thefirst time period. In one aspect, the third time period is at least 24hours after the second time period. In some embodiments, the second andthird time period can be at least 24 hours, at least 48 hours, at least72 hours, and at least 1 week or more after the first or second timeperiod, respectively.

Transcription factors for use in the present invention can be apolypeptide, ribonucleic acid or a nucleic acid. As used herein, theterm “nucleic acid” is intended to include DNA molecules (e.g., cDNA orgenomic DNA), RNA molecules (e.g., mRNA, microRNA or other RNAderivatives), analogs of the DNA or RNA generated using nucleotideanalogs, and derivatives, fragments and homologs thereof. The nucleicacid molecule can be single-stranded or double-stranded. Preferably, thenucleic acid is a DNA.

Preferred transcription factors for use in the methods described hereinare PDX-1, Pax-4, NeuroD1, and MafA. Other transcription factors thatmay be used are Ngn3, and Sox9.

The homeodomain protein PDX-1 (pancreatic and duodenal homeobox gene-1),also known as IDX-1, IPF-1, STF-1, or IUF-1, plays a central role inregulating pancreatic islet development and function. PDX-1 is eitherdirectly or indirectly involved in islet-cell-specific expression ofvarious genes such as, for example insulin, glucagon, somatostatin,proinsulin convertase 1/3 (PC1/3), GLUT-2 and glucokinase. Additionally,PDX-1 mediates insulin gene transcription in response to glucose.Suitable sources of nucleic acids encoding PDX-1 include for example thehuman PDX-1 nucleic acid (and the encoded protein sequences) availableas GenBank Accession Nos. U35632 and AAA88820, respectively. Othersources include rat PDX nucleic acid and protein sequences are shown inGenBank Accession No. U35632 and AAA18355, respectively, and areincorporated herein by reference in their entirety. An additional sourceincludes zebrafish PDX-1 nucleic acid and protein sequences are shown inGenBank Accession No. AF036325 and AAC41260, respectively, and areincorporated herein by reference in their entirety.

Pax-4, also known as paired box 4, paired box protein 4, paired box gene4, MODY9 and KPD, is a pancreatic-specific transcription factor thatbinds to elements within the glucagon, insulin and somatostatinpromoters, and is thought to play an important role in thedifferentiation and development of pancreatic islet beta cells. In somecellular contexts, Pax-4 exhibits repressor activity. Suitable sourcesof nucleic acids encoding Pax-4 include for example the human Pax-4nucleic acid (and the encoded protein sequences) available as GenBankAccession Nos. NM_006193.2 and AAD02289.1, respectively.

MafA, also known as V-maf musculoaponeurotic fibrosarcoma oncogenehomolog A or RIPE3B1, is a beta-cell-specific and glucose-regulatedtranscriptional activator for insulin gene expression. MafA may beinvolved in the function and development of beta-cells as well as in thepathogenesis of diabetes. Suitable sources of nucleic acids encodingMafA include for example the human MafA nucleic acid (and the encodedprotein sequences) available as GenBank Accession Nos. NM_201589.3 andNP_963883.2, respectively.

Neurog3, also known as neurogenin 3 or Ngn3, is a basic helix-loop-helix(bHLH) transcription factor required for endocrine development in thepancreas and intestine. Suitable sources of nucleic acids encodingNeurog3 include for example the human Neurog3 nucleic acid (and theencoded protein sequences) available as GenBank Accession Nos.NM_020999.3 and NP_066279.2, respectively.

NeuroD1, also known as Neuro Differentiation 1, and beta-2 (β2) is aNeuro D-type transcription factor. It is a basic helix-loop-helixtranscription factor that forms heterodimers with other bHLH proteinsand activates transcription of genes that contain a specific DNAsequence known as the E-box. It regulates expression of the insulingene, and mutations in this gene result in type II diabetes mellitus.Suitable sources of nucleic acids encoding NeuroD1 include for examplethe human NeuroD1 nucleic acid (and the encoded protein sequences)available as GenBank Accession Nos. NM_002500.4 and NP_002491.2,respectively.

Sox9 is a transcription factor that is involved in embryonicdevelopment. Sox9 has been particularly investigated for its importancein bone and skeletal development. SOX-9 recognizes the sequence CCTTGAGalong with other members of the HMG-box class DNA-binding proteins. Inthe context of the present invention, the use of Sox9 may be involved inmaintaining the pancreatic progenitor cell mass, either before or afterinduction of transdifferentiation. Suitable sources of nucleic acidsencoding NeuroD1 include for example the human NeuroD1 nucleic acid (andthe encoded protein sequences) available as GenBank Accession Nos.NM_000346.3 and NP_000337.1, respectively.

The cell can be any cell that is capable of producing pancreatichormones, e.g., bone marrow muscle, spleen, kidney, blood, skin,pancreas, or liver. In one embodiment, the cell is a non-pancreaticcell. In one embodiment, the cell is capable of functioning as apancreatic islet cell, i.e., store, process and secrete pancreatichormones, preferably insulin, upon an extracellular trigger. In anotherembodiment the cell is a liver cell. In additional embodiments the cellis totipotent or pluripotent. In alternative embodiments the cell is ahematopoietic stem cell, embryonic stem cell or preferably a hepaticstem cell.

The cell population that is exposed to, i.e., contacted with, thecompounds (i.e. PDX-1, Pax-4, MafA, NeuroD1 and/or Sox-9 polypeptides ornucleic acid encoding PDX-1, Pax-4, MafA, NeuroD1 and/or Sox-9polypeptides) can be any number of cells, i.e., one or more cells, andcan be provided in vitro, in vivo, or ex vivo. The cell population thatis contacted with the transcription factors can be expanded in vitroprior to being contacted with the transcription factors. The cellpopulation produced that exhibits a mature pancreatic beta cellphenotype. These cells can be expanded in vitro by methods known in theart prior to transdifferentiation and maturation along the β-celllineage, and prior to administration or delivery to a patient or subjectin need thereof.

The subject is preferably a mammal. The mammal can be, e.g., a human,non-human primate, mouse, rat, dog, cat, horse, or cow.

In some embodiments, the transcription factor is a polypeptide, such asPDX-1, Pax-4, MafA, NeuroD1 or Sox-9, or combination thereof and isdelivered to a cell by methods known in the art. For example, thetranscription factor polypeptide is provided directly to the cells ordelivered via a microparticle or nanoparticle, e.g., a liposomalcarrier.

In some embodiments, the transcription factor is a nucleic acid. Forexample, the nucleic acid encodes a PDX-1, Pax-4, MafA, NeuroD1 or Sox-9polypeptide. The nucleic acid encoding the transcription factor, or acombination of such nucleic acids, can be delivered to a cell by anymeans known in the art. In some embodiments, the nucleic acid isincorporated in an expression vector or a viral vector. Preferably, theviral vector is an adeno-virus viral vector. The expression or viralvector can be introduced to the cell by any of the following:transfection, electroporation, infection, or transduction.

Cell Populations Predisposed for Transdifferentiation

The present invention provides liver derived cell populations that arepredisposed for transdifferentiation. The cell populations are useful inthe methods of producing pancreatic beta cells described herein. Cellsthat are predisposed for transdifferentiation of the present inventionare also referred to as having increased or enrichedtransdifferentiation capacity. By “increased transdifferentiationcapacity” is meant that when the cell population of the presentinvention is subjected to a differentiation protocol (i.e. introductionof a pancreatic transcription factor), greater than 15%, greater than20%, greater then 30%, greater than 40%, greater than 50%, greater than60%, greater than 70% or greater than 80% will differentiate to analternate cell type. For example, a population of endothelial cells,epithelial cells, mesenchymal cells, fibroblasts, or liver cells withincreased transdifferentiation capacity can be differentiated to maturepancreatic cells or mature neural cells.

In another embodiment, cell populations that are predisposed fortransdifferentation have the capability of activating the glutaminesynthetase response element (GSRE). For example, in the cell populationsof the present invention, at least 2%, at least 3%, at least 4%, atleast 5%, at least 10%, at least 15%, at least 20%, at least 30%, atleast 40%, at least 50%, at least 60%, at least 70%, at least 80% or atleast 90% of the cells in the population are capable of activating GSRE.Preferably, at least 30% of the cells in the population are capable ofactivating GSRE. Glutamine synthetase is an enzyme predominantlyexpressed in the brain, kidneys and liver, and plays an essential rolein the metabolism of nitrogen by catalyzing the condensation ofglutamate and ammonia to form glutamine. Glutamine synthetase is, forexample, uniquely expressed in pericentral liver cells and astrocytes inthe brain. Data presented herein indicate that cells that demonstrateactivation of GSRE provide a unique selective parameter for theisolation of cells predisposed for transdifferentiation.

Activation of GSRE can be measured by methods known to one of ordinaryskill in the art. For example, a recombinant adenovirus can be generatedcontaining the glutamine synthetase response element operatively linkedto a promoter and a reporter gene, such as a fluorescent protein. Thisrecombinant adenovirus with the GSRE-reporter can be introduced into aheterogeneous mixture of cells containing some proportion of cells thatare predisposed for transdifferentiation. Those cells that are competentfor activation of the GSRE will express the reporter gene, which can bedetected by methods known in the art, thereby identifying cellspredisposed for transdifferentiation.

A heterogeneous population of cells, in which those cells predisposedfor transdifferentiation are unknown, can be contacted with anadenoviral vector that contains the GSRE operatively linked to a minimalTK promoter and eGFP. The cells that can activate the GSRE will expressGFP and can be identified by various methods known in the art to detectGFP expression. For example, separation of the GSRE-activated cellswhich are predisposed for transdifferentiation from the cells that arenot predisposed for transdifferentiation can be achieved through FACsapparatus, sorter and techniques known to those ordinarily skilled inthe art (FIG. 14). The separated cells which are predisposed fortransdifferentiation can then be propagated or expanded in vitro.Results described herein demonstrate that passaging of the cellspredisposed for transdifferentiation for 5-12 passages or more retaintheir transdifferentiation capacity. For example, isolated liver cellspredisposed for transdifferentiation continue to produce and secreteinsulin in a glucose-dependent manner even after 12 passages in culture(FIG. 17).

In another embodiment, cell populations that are predisposed fortransdifferentiation also have active Wnt signaling pathways. Wntsignaling pathways play a significant role in stem cell pluripotency andcell fate during development, as well as body axis patterning, cellproliferation, and cell migration. Wnt signaling pathways are activatedby the binding of a Wnt-protein ligand to a Frizzled (Fz) familyreceptor (a G-coupled protein receptor), optionally activating aco-receptor protein, and the subsequent activation of a cytoplasmicprotein called Dishevelled (Dsh). In the canonical Wnt pathway,co-receptor LRP-5/6 is also activated and beta-catenin accumulates inthe cytoplasm and is eventually translocated into the nucleus to act asa transcriptional coactivator of TCF/LEF transcription factors. WithoutWnt signaling, a destruction complex which includes proteinsadenomatosis polyposis coli (APC), Axin, protein phosphatase 2A (PP2A),glycogen synthase kinase 3 (GSK3) and casein kinase 1α (CK1α) targetsβ-catenin for ubiquitination and its subsequent degradation by theproteasome. However, activation of the Frizzled receptor by Wnt bindingcauses disruption of the destruction complex, thereby allowing β-cateninto accumulate.

Wnt signaling can also occur through noncanonical pathways that utilizedifferent co-receptor proteins and activate different downstreameffectors to, for example, regulate of the cytoskeleton, stimulate ofcalcium release from the endoplasmic reticulum, activate mTOR pathways,and regulate myogenesis.

One of ordinary skill in the art could readily use methods known in theart to determine the activation of Wnt signaling pathways. For example,cells that express Wnt3a, decreased levels of DKK1 or DKK3, decreasedlevels of APC, increased activated beta-catenin levels, or STAT3 bindingelements have active Wnt signaling pathways. DKK1, DKK3, and APC areknown inhibitors of Wnt signaling pathways. Other signaling effectorsthat indicate active Wnt signaling pathways are readily known in theart.

Preferably, the cell populations are predisposed fortransdifferentiation to the pancreatic lineage, wherein thetransdifferentiated cells exhibit pancreatic phenotype and function. Forexample, the transdifferentiated cells exhibit mature pancreatic betacell phenotype and function, which includes, but is not limited to,expression, production, and/or secretion of pancreatic hormones.Pancreatic hormones can include, but are not limited to, insulin,somatostatin, glucagon, or islet amyloid polypeptide (IAPP). Insulin canbe hepatic insulin or serum insulin. Preferably the insulin is a fullyprocess form of insulin capable of promoting flucose utilization, andcarbohydrate, fat and protein metabolism. For example, the cellspredisposed for transdifferentiation may encompass about 15% of all thecells in a heterogeneous in vitro primary human liver cell culture. Whenthe cells ectopically express pTFs, greater than 5%, 10%, 15%, 20%, 25%,30%, 40%, 50% of the cells in culture produce insulin or secretec-peptide.

In one embodiment, cell populations that are predisposed fortransdifferentiation are located in close proximity to the central veinsof the liver, or are pericentral liver cells. As shown herein, althoughover 40-50% of liver cells that ectopically express pancreatictranscription factors, such as PDX-1, only a subset of cells producedinsulin upon pTF expression. These insulin-producing cells (IPCs) wereprimarily located close to the ventral veins, as shown by FIG. 1B. Thesecells are also characterized by expression of glutamine synthetase andactive Wnt signaling.

In another preferred embodiment, the cell populations of the presentinvention are predisposed for transdifferentiation to the neurallineage, wherein the transdifferentiated cells express neural markers,exhibit neural phenotype, or exhibit neural function. Thetransdifferentiated cells can be neurons or glial cells.

In another embodiment, cells with increased predisposition fortransdifferentiation may be identified through specific cell surfacemarkers. For example, cells with increased levels of HOMER1, LAMP3 orBMPR2 indicate cells with increased transdifferentiation capacity whencompared to cells without predisposition for transdifferentiation. Cellswith decreased levels of ABCB1, ITGA4, ABCB4, or PRNP indicate cellswith increased transdifferentiation capacity when compared to cellswithout predisposition for transdifferentiation. Any combination of thecell surface markers described can be used to distinguish a cellpopulation predisposed to transdifferentiation from a cell populationthat is not predisposed to transdifferentiation. Antibodies to thesecell surface markers are commercially available Immuno-assay orimmune-affinity techniques known in the art may be utilized todistinguish cells with increased transdifferentiation capacity fromthose cells without transdifferentiation capacity.

Use of the cell populations of the present invention to produce cellsthat exhibit pancreatic cell phenotypes provide certain advantages overdifferentiating heterogeneous populations of non-pancreatic cells toproduce cells that exhibit pancreatic cell phenotypes. Previous studiesthat describe expressing a pancreatic transcription factor (pTF) such asPDX-1 in a heterogeneous population of non-pancreatic cells (i.e., livercells) show that at best, only 15% of the PDX-1-expressing cells arecompetent for producing insulin. Therefore, only 15% of the cells weresuccessfully differentiated to a mature pancreatic beta-cell capable ofproducing and secreting pancreatic hormones. In contrast, introducingpTFs into the cell populations of the present invention results in atleast 30%, at least 40%, at least 50%, at least 60%, at least 70%, or atleast 80% of the cells are differentiated to a mature pancreatic betacell phenotype, for example, produce insulin, glucagon, and/or secretec-peptide. Preferably, when the cells of the cell population of thepresent invention express a pancreatic transcription factor, at least30% of the cells produce insulin or secrete c-peptide.

Methods of Transdifferentiation

The present invention also provides methods for utilizing the cellpopulations with increased transdifferentiation capacity to producecells that exhibit a mature differentiated cell type, where thedifferentiated cell has a different phenotype from the starting cellpopulation. For example, a population of cells with increasedtransdifferentiation capacity (i.e. epithelial cells, fibroblasts orliver cells) can be differentiated to cells along the pancreatic orneural lineage to exhibit mature differentiated pancreatic or neuralcell phenotypes. Any means known in the art for differentiating cells topancreatic or neural lineage can be utilized.

In one embodiment, the cell population predisposed fortransdifferentiated may be differentiated along the neural lineagethrough the expression of neural transcription factors. Suitable neuraltranscription factors are known in the art. In other embodiments, thecell population of the present invention may be differentiated to matureneural cells through contacting the cells with various cytokines, growthfactors, or other agents known in the art to differentiate cells to theneural lineage. The differentiated neural cells may express neuralmarkers, exhibit a neural phenotype (i.e., neural gene expressionprofile), or exhibit neural function. The differentiated cells can beneurons or glial cells.

In another embodiment, the cell population predisposed fortransdifferentation may be differentiated along the pancreatic lineagethrough the expression of pancreatic transcription factors. Thepancreatic transcription factors are, for example, PDX-1, Pax-4, MafA,NeuroD1, or a combination thereof. Methods for producing pancreatic betacells are described in U.S. Pat. No. 6,774,120 and U.S. Publication No.2005/0090465, the contents of which are incorporated by reference intheir entireties.

In another embodiment, the cell population predisposed fortransdifferentation may be differentiated along the pancreatic lineagethrough the methods described herein.

Pancreatic Beta-Cell Phenotypes

The methods provided herein produce cells with a mature pancreatic betacell phenotype or function. By “pancreatic beta cell phenotype orfunction” is meant that the cell displays one or more characteristicstypical of pancreatic beta cells, i.e. pancreatic hormone production,processing, storage in secretory granules, hormone secretion, activationof pancreatic gene promoters, or characteristic beta cell geneexpression profile. Hormone secretion includes nutritionally and/orhormonally regulated secretion. Preferably, the cells produced exhibitat least one pancreatic beta cell phenotype or function, as describedherein.

The pancreatic hormone can be for example, insulin, glucagon,somatostatin or islet amyloid polypeptide (IAPP). Insulin can be hepaticinsulin or serum insulin. In another embodiment the pancreatic hormoneis hepatic insulin. In an alternative embodiment the pancreatic hormoneis serum insulin (i.e., a fully processed form of insulin capable ofpromoting, e.g., glucose utilization, carbohydrate, fat and proteinmetabolism).

In some embodiments the pancreatic hormone is in the “prohormone” form.In other embodiments the pancreatic hormone is in the fully processedbiologically active form of the hormone. In other embodiments thepancreatic hormone is under regulatory control i.e., secretion of thehormone is under nutritional and hormonal control similar toendogenously produced pancreatic hormones. For example, in one aspect ofthe invention the hormone is under the regulatory control of glucose.Insulin secretion can also be measured by, for example, C-peptideprocessing and secretion.

The pancreatic beta cell phenotype can be determined for example bymeasuring pancreatic hormone production, i.e., insulin, somatostatin orglucagon protein mRNA or protein expression. Hormone production can bedetermined by methods known in the art, i.e. immunoassay, western blot,receptor binding assays or functionally by the ability to amelioratehyperglycemia upon implantation in a diabetic host.

In some embodiments, the cells can be directed to produce and secreteinsulin using the methods specified herein. The ability of a cell toproduce insulin can be assayed by a variety of methods known to those ofordinary skill in the art. For example, insulin mRNA can be detected byRT-PCR or insulin may be detected by antibodies raised against insulin.In addition, other indicators of pancreatic differentiation include theexpression of the genes Isl-1, Pdx-1, Pax-4, Pax-6, and Glut-2. Otherphenotypic markers for the identification of islet cells are disclosedin U.S. 2003/0138948, incorporated herein in its entirety.

The pancreatic beta cell phenotype can be determined for example bypromoter activation of pancreas-specific genes. Pancreas-specificpromoters of particular interest include the promoters for insulin andpancreatic transcription factors, i.e. endogenous PDX-1. Promoteractivation can be determined by methods known in the art, for example byluciferase assay, EMSA, or detection of downstream gene expression.

In some embodiments, the pancreatic beta-cell phenotype can also bedetermined by induction of a pancreatic gene expression profile. By“pancreatic gene expression profile” it is meant: to include expressionof one or more genes that are normally transcriptionally silent innon-endocrine tissues, i.e., a pancreatic transcription factor,pancreatic enzymes or pancreatic hormones. Pancreatic enzymes are, forexample, PCSK2 (PC2 or prohormone convertase), PC1/3 (prohormoneconvertase 1/3), glucokinase, glucose transporter 2 (GLUT-2).Pancreatic-specific transcription factors include, for example, Nkx2.2,Nkx6.1, Pax-4, Pax-6, MafA, NeuroD1, NeuroG3, Ngn3, beta-2, ARX, BRAIN4and Isl-1.

Induction of the pancreatic gene expression profile can be detectedusing techniques well known to one of ordinary skill in the art. Forexample, pancreatic hormone RNA sequences can be detected in, e.g.,northern blot hybridization analyses, amplification-based detectionmethods such as reverse-transcription based polymerase chain reaction orsystemic detection by microarray chip analysis. Alternatively,expression can be also measured at the protein level, i.e., by measuringthe levels of polypeptides encoded by the gene. In a specific embodimentPC1/3 gene or protein expression can be determined by its activity inprocessing prohormones to their active mature form. Such methods arewell known in the art and include, e.g., immunoassays based onantibodies to proteins encoded by the genes, or HPLC of the processedprohormones.

In some embodiments, the cells exhibiting a mature beta-cell phenotypegenerated by the methods described herein may repress at least one geneor the gene expression profile of the original cell. For example, aliver cell that is induced to exhibit a mature beta-cell phenotype mayrepress at least one liver-specific gene. One skilled in the art couldreadily determine the liver-specific gene expression of the originalcell and the produced cells using methods known in the art, i.e.measuring the levels of mRNA or polypeptides encoded by the genes. Uponcomparison, a decrease in the liver-specific gene expression wouldindicate that transdifferentiation has occurred.

Methods of Treating a Pancreatic Disorder

The present invention discloses methods for use in treating, i.e.,preventing or delaying the onset or alleviating a symptom of apancreatic disorder in a subject. For example, the pancreatic disorderis a degenerative pancreatic disorder. The methods disclosed herein areparticularly useful for those pancreatic disorders that are caused by orresult in a loss of pancreatic cells, e.g., islet beta-cells, or a lossin pancreatic cell function.

Common degenerative pancreatic disorders include, but are not limitedto: diabetes (e.g., type I, type II, or gestational) and pancreaticcancer. Other pancreatic disorders or pancreas-related disorders thatmay be treated by using the methods disclosed herein are, for example,hyperglycemia, pancreatitis, pancreatic pseudocysts or pancreatic traumacaused by injury.

Diabetes is a metabolic disorder found in three forms: type 1, type 2and gestational. Type 1, or IDDM, is an autoimmune disease; the immunesystem destroys the pancreas' insulin-producing beta cells, reducing oreliminating the pancreas' ability to produce insulin. Type 1 diabetespatients must take daily insulin supplements to sustain life. Symptomstypically develop quickly and include increased thirst and urination,chronic hunger, weight loss, blurred vision and fatigue. Type 2 diabetesis the most common, found in 90 percent to 95 percent of diabetessufferers. It is associated with older age, obesity, family history,previous gestational diabetes, physical inactivity and ethnicity.Gestational diabetes occurs only in pregnancy. Women who developgestational diabetes have a 20 percent to 50 percent chance ofdeveloping type 2 diabetes within five to 10 years.

A subject suffering from or at risk of developing diabetes is identifiedby methods known in the art such as determining blood glucose levels.For example, a blood glucose value above 140 mg/dL on at least twooccasions after an overnight fast means a person has diabetes. A personnot suffering from or at risk of developing diabetes is characterized ashaving fasting sugar levels between 70-110 mg/dL.

Symptoms of diabetes include fatigue, nausea, frequent urination,excessive thirst, weight loss, blurred vision, frequent infections andslow healing of wounds or sores, blood pressure consistently at or above140/90, HDL cholesterol less than 35 mg/dL or triglycerides greater than250 mg/dL, hyperglycemia, hypoglycemia, insulin deficiency orresistance. Diabetic or pre-diabetic patients to which the compounds areadministered are identified using diagnostic methods know in the art.

Hyperglycemia is a pancreas-related disorder in which an excessiveamount of glucose circulates in the blood plasma. This is generally aglucose level higher than (200 mg/dl). A subject with hyperglycemia mayor may not have diabetes.

Pancreatic cancer is the fourth most common cancer in the U.S., mainlyoccurs in people over the age of 60, and has the lowest five-yearsurvival rate of any cancer. Adenocarcinoma, the most common type ofpancreatic cancer, occurs in the lining of the pancreatic duct;cystadenocarcinoma and acinar cell carcinoma are rarer. However, benigntumors also grow within the pancreas; these include insulinoma—a tumorthat secretes insulin, gastrinoma—which secretes higher-than-normallevels of gastrin, and glucagonoma—a tumor that secretes glucagon.

Pancreatic cancer has no known causes, but several risks, includingdiabetes, cigarette smoking and chronic pancreatitis. Symptoms mayinclude upper abdominal pain, poor appetite, jaundice, weight loss,indigestion, nausea or vomiting, diarrhea, fatigue, itching or enlargedabdominal organs. Diagnosis is made using ultrasound, computedtomography scan, magnetic resonance imaging, ERCP, percutaneoustranshepatic cholangiography, pancreas biopsy or blood tests. Treatmentmay involve surgery, radiation therapy or chemotherapy, medication forpain or itching, oral enzymes preparations or insulin treatment.

Pancreatitis is the inflammation and autodigestion of the pancreas. Inautodigestion, the pancreas is destroyed by its own enzymes, which causeinflammation. Acute pancreatitis typically involves only a singleincidence, after which the pancreas will return to normal. Chronicpancreatitis, however, involves permanent damage to the pancreas andpancreatic function and can lead to fibrosis. Alternately, it mayresolve after several attacks. Pancreatis is most frequently caused bygallstones blocking the pancreatic duct or by alcohol abuse, which cancause the small pancreatic ductules to be blocked. Other causes includeabdominal trauma or surgery, infections, kidney failure, lupus, cysticfibrosis, a tumor or a scorpion's venomous sting.

Symptoms frequently associated with pancreatitis include abdominal pain,possibly radiating to the back or chest, nausea or vomiting, rapidpulse, fever, upper abdominal swelling, ascites, lowered blood pressureor mild jaundice. Symptoms may be attributed to other maladies beforebeing identified as associated with pancreatitis.

Method of Treating a Neurological Disorders

The present invention also provides methods for treating a subject witha neurological disease or disorder, such as a neurodegenerative diseasedisorder. The population of cells described herein is useful fortreating a subject with a neurological disease or disorder that ischaracterized by loss of neural cells or neural function, by way ofreplenishing the degenerated or nonfunctional cells. Neurodegenerativediseases that may be treated using the methods described herein include,but are not limited to, Parkinson's disease, Parkinsonian disorders,Alzheimer's disease, Huntington's disease, amyotrophic lateralsclerosis, Lewy body disease, age-related neurodegeneration,neurological cancers, and brain trauma resulting from surgery, accident,ischemia, or stroke. The population of cells described herein can bedifferentiated to a neural cell population with neural function, and thedifferentiated neural cell population may be administered to a subjectwith a neurological disease or disorder.

Therapeutics Compositions

The herein-described transdifferentiation-inducing compounds, or ectopicpancreatic transcription factors (i.e., PDX-1, Pax-4, MafA, NeuroD1 orSox-9 polypeptides, ribonucleic acids or nucleic acids encoding PDX-1,Pax-4, MafA, NeuroD1 or Sox-9 polypeptides), when used therapeutically,are referred to herein as “Therapeutics”. Methods of administration ofTherapeutics include, but are not limited to, intradermal,intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal,epidural, and oral routes. The Therapeutics of the present invention maybe administered by any convenient route, for example by infusion orbolus injection, by absorption through epithelial or mucocutaneouslinings (e.g., oral mucosa, rectal and intestinal mucosa, etc.) and maybe administered together with other biologically-active agents.Administration can be systemic or local. In addition, it may beadvantageous to administer the Therapeutic into the central nervoussystem by any suitable route, including intraventricular and intrathecalinjection. Intraventricular injection may be facilitated by anintraventricular catheter attached to a reservoir (e.g., an Ommayareservoir) Pulmonary administration may also be employed by use of aninhaler or nebulizer, and formulation with an aerosolizing agent. It mayalso be desirable to administer the Therapeutic locally to the area inneed of treatment; this may be achieved by, for example, and not by wayof limitation, local infusion during surgery, topical application, byinjection, by means of a catheter, by means of a suppository, or bymeans of an implant. Various delivery systems are known and can be usedto administer a Therapeutic of the present invention including, e.g.:(i) encapsulation in liposomes, microparticles, microcapsules; (ii)recombinant cells capable of expressing the Therapeutic; (iii)receptor-mediated endocytosis (See, e.g., Wu and Wu, 1987. J Biol Chem262:4429-4432); (iv) construction of a Therapeutic nucleic acid as partof a retroviral, adenoviral or other vector, and the like. In oneembodiment of the present invention, the Therapeutic may be delivered ina vesicle, in particular a liposome. In a liposome, the protein of thepresent invention is combined, in addition to other pharmaceuticallyacceptable carriers, with amphipathic agents such as lipids which existin aggregated form as micelles, insoluble monolayers, liquid crystals,or lamellar layers in aqueous solution. Suitable lipids for liposomalformulation include, without limitation, monoglycerides, diglycerides,sulfatides, lysolecithin, phospholipids, saponin, bile acids, and thelike. Preparation of such liposomal formulations is within the level ofskill in the art, as disclosed, for example, in U.S. Pat. No. 4,837,028;and U.S. Pat. No. 4,737,323, all of which are incorporated herein byreference. In yet another embodiment, the Therapeutic can be deliveredin a controlled release system including, e.g.: a delivery pump (See,e.g., Saudek, et al., 1989. New Engl J Med 321:574 and a semi-permeablepolymeric material (See, e.g., Howard, et al., 1989. J Neurosurg71:105). Additionally, the controlled release system can be placed inproximity of the therapeutic target (e.g., the brain), thus requiringonly a fraction of the systemic dose. See, e.g., Goodson, In: MedicalApplications of Controlled Release 1984. (CRC Press, Boca Raton, Fla.).

In a specific embodiment of the present invention, where the Therapeuticis a nucleic acid encoding a protein, the Therapeutic nucleic acid maybe administered in vivo to promote expression of its encoded protein, byconstructing it as part of an appropriate nucleic acid expression vectorand administering it so that it becomes intracellular (e.g., by use of aretroviral vector, by direct injection, by use of microparticlebombardment, by coating with lipids or cell-surface receptors ortransfecting agents, or by administering it in linkage to ahomeobox-like peptide which is known to enter the nucleus (See, e.g.,Joliot, et al., 1991. Proc Natl Acad Sci USA 88:1864-1868), and thelike. Alternatively, a nucleic acid Therapeutic can be introducedintracellularly and incorporated within host cell DNA for expression, byhomologous recombination or remain episomal.

Preferably, the Therapeutic is administered intravenously. Specifically,the Therapeutic can be delivered via a portal vein infusion.

As used herein, the term “therapeutically effective amount” means thetotal amount of each active component of the pharmaceutical compositionor method that is sufficient to show a meaningful patient benefit, i.e.,treatment, healing, prevention or amelioration of the relevant medicalcondition, or an increase in rate of treatment, healing, prevention oramelioration of such conditions. When applied to an individual activeingredient, administered alone, the term refers to that ingredientalone. When applied to a combination, the term refers to combinedamounts of the active ingredients that result in the therapeutic effect,whether administered in combination, serially or simultaneously.

The amount of the Therapeutic of the invention which will be effectivein the treatment of a particular disorder or condition will depend onthe nature of the disorder or condition, and may be determined bystandard clinical techniques by those of average skill within the art.In addition, in vitro assays may optionally be employed to help identifyoptimal dosage ranges. The precise dose to be employed in theformulation will also depend on the route of administration, and theoverall seriousness of the disease or disorder, and should be decidedaccording to the judgment of the practitioner and each patient'scircumstances. Ultimately, the attending physician will decide theamount of protein of the present invention with which to treat eachindividual patient. Initially, the attending physician will administerlow doses of protein of the present invention and observe the patient'sresponse. Larger doses of protein of the present invention may beadministered until the optimal therapeutic effect is obtained for thepatient, and at that point the dosage is not increased further. However,suitable dosage ranges for intravenous administration of theTherapeutics of the present invention are generally at least 1 milliontransdifferentiated cells, at least 2 million transdifferentiated cells,at least 5 million transdifferentiated cells, at least 10 milliontransdifferentiated cells, at least 25 million transdifferentiatedcells, at least 50 million transdifferentiated cells, at least 100million transdifferentiated cells, at least 200 milliontransdifferentiated cells, at least 300 million transdifferentiatedcells, at least 400 million transdifferentiated cells, at least 500million transdifferentiated cells, at least 600 milliontransdifferentiated cells, at least 700 million transdifferentiatedcells, at least 800 million transdifferentiated cells, at least 900million transdifferentiated cells, at least 1 billiontransdifferentiated cells, at least 2 billion transdifferentiated cells,at least 3 billion transdifferentiated cells, at least 4 billiontransdifferentiated cells, or at least 5 billion transdifferentiatedcells. Preferably, the dose is 1-2 billion transdifferentiated cellsinto a 60-75 kg subject. One skilled in the art would appreciate thateffective doses may be extrapolated from dose-response curves derivedfrom in vitro or animal model test systems.

The duration of intravenous therapy using the Therapeutic of the presentinvention will vary, depending on the severity of the disease beingtreated and the condition and potential idiosyncratic response of eachindividual patient. It is contemplated that the duration of eachapplication of the protein of the present invention will be in the rangeof 12 to 24 hours of continuous intravenous administration. Ultimatelythe attending physician will decide on the appropriate duration oftherapy using the pharmaceutical composition of the present invention.

Cells may also be cultured ex vivo in the presence of therapeuticagents, nucleic acids, or proteins of the present invention in order toproliferate or to produce a desired effect on or activity in such cells.Treated cells can then be introduced in vivo via the administrationroutes described herein for therapeutic purposes.

Recombinant Expression Vectors and Host Cells

Another aspect of the invention pertains to vectors, preferablyexpression vectors, containing a nucleic acid encoding a PDX, Pax-4,NeuroD1 or MafA protein, or other pancreatic transcription factor, suchas Ngn3, or derivatives, fragments, analogs, homologs or combinationsthereof. In some embodiments, the expression vector comprises a singlenucleic acid encoding any of the following transcription factors: PDX-1,Pax-4, NeuroD1, Ngn3, MafA, or Sox-9 or derivatives or fragmentsthereof. In some embodiments, the expression vector comprises twonucleic acids encoding any combination of the following transcriptionfactors: PDX-1, Pax-4, NeuroD1, Ngn3, MafA, or Sox-9 or derivatives orfragments thereof. In a preferred embodiment, the expression vectorcontains nucleic acids encoding PDX-1 and NeuroD1.

As used herein, the term “vector” refers to a nucleic acid moleculecapable of transporting another nucleic acid to which it has beenlinked. One type of vector is a “plasmid”, which refers to a linear orcircular double stranded DNA loop into which additional DNA segments canbe ligated. Another type of vector is a viral vector, wherein additionalDNA segments can be ligated into the viral genome. Certain vectors arecapable of autonomous replication in a host cell into which they areintroduced (e.g., bacterial vectors having a bacterial origin ofreplication and episomal mammalian vectors). Other vectors (e.g.,non-episomal mammalian vectors) are integrated into the genome of a hostcell upon introduction into the host cell, and thereby are replicatedalong with the host genome. Moreover, certain vectors are capable ofdirecting the expression of genes to which they are operatively linked.Such vectors are referred to herein as “expression vectors”. In general,expression vectors of utility in recombinant DNA techniques are often inthe form of plasmids. In the present specification, “plasmid” and“vector” can be used interchangeably as the plasmid is the most commonlyused form of vector. However, the invention is intended to include suchother forms of expression vectors, such as viral vectors (e.g.,replication defective retroviruses, lentivirus, adenoviruses andadeno-associated viruses), which serve equivalent functions.Additionally, some viral vectors are capable of targeting a particularcells type either specifically or non-specifically.

The recombinant expression vectors of the invention comprise a nucleicacid of the invention in a form suitable for expression of the nucleicacid in a host cell, which means that the recombinant expression vectorsinclude one or more regulatory sequences, selected on the basis of thehost cells to be used for expression, that is operatively linked to thenucleic acid sequence to be expressed. Within a recombinant expressionvector, “operably linked” is intended to mean that the nucleotidesequence of interest is linked to the regulatory sequence(s) in a mannerthat allows for expression of the nucleotide sequence (e.g., in an invitro transcription/translation system or in a host cell when the vectoris introduced into the host cell). The term “regulatory sequence” isintended to include promoters, enhancers and other expression controlelements (e.g., polyadenylation signals). Such regulatory sequences aredescribed, for example, in Goeddel; GENE EXPRESSION TECHNOLOGY: METHODSIN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990). Regulatorysequences include those that direct constitutive expression of anucleotide sequence in many types of host cell and those that directexpression of the nucleotide sequence only in certain host cells (e.g.,tissue-specific regulatory sequences). It will be appreciated by thoseskilled in the art that the design of the expression vector can dependon such factors as the choice of the host cell to be transformed, thelevel of expression of protein desired, etc. The expression vectors ofthe invention can be introduced into host cells to thereby produceproteins or peptides, including fusion proteins or peptides, encoded bynucleic acids as described herein (e.g., PDX-1, Pax-4, MafA, NeuroD1 orSox-9 proteins, or mutant forms or fusion proteins thereof, etc.).

For example, an expression vector comprises one nucleic acid encoding atranscription factor operably linked to a promoter. In expressionvectors comprising two nucleic acids encoding transcription factors,each nucleic acid may be operably linked to a promoter. The promoteroperably linked to each nucleic acid may be different or the same.Alternatively, the two nucleic acids may be operably linked to a singlepromoter. Promoters useful for the expression vectors of the inventioncan be any promoter known in the art. The ordinarily skilled artisancould readily determine suitable promoters for the host cell in whichthe nucleic acid is to be expressed, the level of expression of proteindesired, or the timing of expression, etc. The promoter may be aconstitutive promoter, an inducible promoter, or a cell-type specificpromoter.

The recombinant expression vectors of the invention can be designed forexpression of PDX-1 in prokaryotic or eukaryotic cells. For example,PDX-1, Pax-4, MafA, NeuroD1, and/or Sox-9 can be expressed in bacterialcells such as E. coli, insect cells (using baculovirus expressionvectors) yeast cells or mammalian cells. Suitable host cells arediscussed further in Goeddel, GENE EXPRESSION TECHNOLOGY: METHODS INENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990). Alternatively,the recombinant expression vector can be transcribed and translated invitro, for example using T7 promoter regulatory sequences and T7polymerase.

Expression of proteins in prokaryotes is most often carried out in E.coli with vectors containing constitutive or inducible promotersdirecting the expression of either fusion or non-fusion proteins. Fusionvectors add a number of amino acids to a protein encoded therein,usually to the amino terminus of the recombinant protein. Such fusionvectors typically serve three purposes: (1) to increase expression ofrecombinant protein; (2) to increase the solubility of the recombinantprotein; and (3) to aid in the purification of the recombinant proteinby acting as a ligand in affinity purification. Often, in fusionexpression vectors, a proteolytic cleavage site is introduced at thejunction of the fusion moiety and the recombinant protein to enableseparation of the recombinant protein from the fusion moiety subsequentto purification of the fusion protein. Such enzymes, and their cognaterecognition sequences, include Factor Xa, thrombin and enterokinase.Typical fusion expression vectors include pGEX (Pharmacia Biotech Inc;Smith and Johnson (1988) Gene 67:31-40), pMAL (New England Biolabs,Beverly, Mass.) and pRITS (Pharmacia, Piscataway, N.J.) that fuseglutathione S-transferase (GST), maltose E binding protein, or proteinA, respectively, to the target recombinant protein.

Examples of suitable inducible non-fusion E. coli expression vectorsinclude pTrc (Amrann et al., (1988) Gene 69:301-315) and pET 11d(Studier et al., GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185,Academic Press, San Diego, Calif. (1990) 60-89).

One strategy to maximize recombinant protein expression in E. coli is toexpress the protein in host bacteria with an impaired capacity toproteolytically cleave the recombinant protein. See, Gottesman, GENEEXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, SanDiego, Calif. (1990) 119-128. Another strategy is to alter the nucleicacid sequence of the nucleic acid to be inserted into an expressionvector so that the individual codons for each amino acid are thosepreferentially utilized in E. coli (Wada et al., (1992) Nucleic AcidsRes. 20:2111-2118). Such alteration of nucleic acid sequences of theinvention can be carried out by standard DNA synthesis techniques.

In another embodiment, the PDX-1, Pax-4, MafA, NeuroD1, or Sox-9expression vector is a yeast expression vector. Examples of vectors forexpression in yeast S. cerevisiae include pYepSec1 (Baldari, et al.,(1987) EMBO J 6:229-234), pMFa (Kujan and Herskowitz, (1982) Cell30:933-943), pJRY88 (Schultz et al., (1987) Gene 54:113-123), pYES2(Invitrogen Corporation, San Diego, Calif.), and picZ (InVitrogen Corp,San Diego, Calif.).

Alternatively, PDX-1, Pax-4, MafA, NeuroD1 or Sox-9 can be expressed ininsect cells using baculovirus expression vectors. Baculovirus vectorsavailable for expression of proteins in cultured insect cells (e.g., SF9cells) include the pAc series (Smith et al. (1983) Mol Cell Biol3:2156-2165) and the pVL series (Lucklow and Summers (1989) Virology170:31-39).

In yet another embodiment, a nucleic acid of the invention is expressedin mammalian cells using a mammalian expression vector. Examples ofmammalian expression vectors include pCDM8 (Seed (1987) Nature 329:840)and pMT2PC (Kaufman et al. (1987) EMBO J 6: 187-195). When used inmammalian cells, the expression vector's control functions are oftenprovided by viral regulatory elements. For example, commonly usedpromoters are derived from polyoma, Adenovirus 2, cytomegalovirus andSimian Virus 40. For other suitable expression systems for bothprokaryotic and eukaryotic cells. See, e.g., Chapters 16 and 17 ofSambrook et al., MOLECULAR CLONING: A LABORATORY MANUAL. 2nd ed., ColdSpring Harbor Laboratory, Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N. Y., 1989.

In another embodiment, the recombinant mammalian expression vector iscapable of directing expression of the nucleic acid preferentially in aparticular cell type (e.g., tissue-specific regulatory elements are usedto express the nucleic acid). Tissue-specific regulatory elements areknown in the art. Non-limiting examples of suitable tissue-specificpromoters include the albumin promoter (liver-specific; Pinkert et al.(1987) Genes Dev 1:268-277), lymphoid-specific promoters (Calame andEaton (1988) Adv Immunol 43:235-275), in particular promoters of T cellreceptors (Winoto and Baltimore (1989) EMBO J 8:729-733) andimmunoglobulins (Banerji et al. (1983) Cell 33:729-740; Queen andBaltimore (1983) Cell 33:741-748), neuron-specific promoters (e.g., theneurofilament promoter; Byrne and Ruddle (1989) PNAS 86:5473-5477),pancreas-specific promoters (Edlund et al. (1985) Science 230:912-916),and mammary gland-specific promoters (e.g., milk whey promoter; U.S.Pat. No. 4,873,316 and European Application Publication No. 264,166).Developmentally-regulated promoters are also encompassed, e.g., themurine hox promoters (Kessel and Gruss (1990) Science 249:374-379) andthe alpha-fetoprotein promoter (Campes and Tilghman (1989) Genes Dev3:537-546).

The invention further provides a recombinant expression vectorcomprising a DNA molecule of the invention cloned into the expressionvector in an antisense orientation. That is, the DNA molecule isoperatively linked to a regulatory sequence in a manner that allows forexpression (by transcription of the DNA molecule) of an RNA moleculethat is antisense to PDX mRNA. Regulatory sequences operatively linkedto a nucleic acid cloned in the antisense orientation can be chosen thatdirect the continuous expression of the antisense RNA molecule in avariety of cell types, for instance viral promoters and/or enhancers, orregulatory sequences can be chosen that direct constitutive, tissuespecific or cell type specific expression of antisense RNA. Theantisense expression vector can be in the form of a recombinant plasmid,phagemid or attenuated virus in which antisense nucleic acids areproduced under the control of a high efficiency regulatory region, theactivity of which can be determined by the cell type into which thevector is introduced. For a discussion of the regulation of geneexpression using antisense genes see Weintraub et al., “Antisense RNA asa molecular tool for genetic analysis,” Reviews—Trends in Genetics, Vol.1(1) 1986.

Another aspect of the invention pertains to host cells into which arecombinant expression vector of the invention has been introduced. Theterms “host cell” and “recombinant host cell” are used interchangeablyherein. It is understood that such terms refer not only to theparticular subject cell but also to the progeny or potential progeny ofsuch a cell. Because certain modifications may occur in succeedinggenerations due to either mutation or environmental influences, suchprogeny may not, in fact, be identical to the parent cell, but are stillincluded within the scope of the term as used herein. Additionally, hostcells could be modulated once expressing PDX-1, Pax-4, MafA, NeuroD1 orSox-9 or a combination thereof, and may either maintain or looseoriginal characteristics.

A host cell can be any prokaryotic or eukaryotic cell. For example,PDX-1, Pax-4, MafA, NeuroD1 or Sox-9 protein can be expressed inbacterial cells such as E. coli, insect cells, yeast or mammalian cells(such as Chinese hamster ovary cells (CHO) or COS cells). Alternatively,a host cell can be a premature mammalian cell, i.e., pluripotent stemcell. A host cell can also be derived from other human tissue. Othersuitable host cells are known to those skilled in the art.

Vector DNA can be introduced into prokaryotic or eukaryotic cells viaconventional transformation, transduction, infection or transfectiontechniques. As used herein, the terms “transformation” “transduction”,“infection” and “transfection” are intended to refer to a variety ofart-recognized techniques for introducing foreign nucleic acid (e.g.,DNA) into a host cell, including calcium phosphate or calcium chlorideco-precipitation, DEAE-dextran-mediated transfection, lipofection, orelectroporation. In addition transfection can be mediated by atransfection agent. By “transfection agent” is meant to include anycompound that mediates incorporation of DNA in the host cell, e.g.,liposome. Suitable methods for transforming or transfecting host cellscan be found in Sambrook, et al. (MOLECULAR CLONING: A LABORATORYMANUAL. 2nd ed., Cold Spring Harbor Laboratory, Cold Spring HarborLaboratory Press, Cold Spring Harbor, N. Y., 1989), and other laboratorymanuals.

Transfection may be “stable” (i.e. integration of the foreign DNA intothe host genome) or “transient” (i.e., DNA is episomally expressed inthe host cells).

For stable transfection of mammalian cells, it is known that, dependingupon the expression vector and transfection technique used, only a smallfraction of cells may integrate the foreign DNA into their genome theremainder of the DNA remains episomal In order to identify and selectthese integrants, a gene that encodes a selectable marker (e.g.,resistance to antibiotics) is generally introduced into the host cellsalong with the gene of interest. Various selectable markers includethose that confer resistance to drugs, such as G418, hygromycin andmethotrexate. Nucleic acid encoding a selectable marker can beintroduced into a host cell on the same vector as that encoding PDX orcan be introduced on a separate vector. Cells stably transfected withthe introduced nucleic acid can be identified by drug selection (e.g.,cells that have incorporated the selectable marker gene will survive,while the other cells die). In another embodiment the cells modulated byPDX or the transfected cells are identified by the induction ofexpression of an endogenous reporter gene. In a specific embodiment, thepromoter is the insulin promoter driving the expression of greenfluorescent protein (GFP).

In one embodiment the PDX-1, Pax-4, MafA, NeuroD1, or Sox-9 nucleic acidis present in a viral vector. In one embodiment, the PDX-1 and NeuroD1nucleic acids are present in the same viral vector. In anotherembodiment the PDX-1, Pax-4, MafA, NeuroD1, or Sox-9 nucleic acid isencapsulated in a virus. In another embodiment, the PDX-1 and NeuroD1 isencapsulated in a virus (i.e., nucleic acids encoding PDX-1 and NeuroD1are encapsulated in the same virus particle). In some embodiments thevirus preferably infects pluripotent cells of various tissue type, e.g.hematopoietic stem, cells, neuronal stem cells, hepatic stem cells orembryonic stem cells, preferably the virus is hepatotropic. By“hepatotropic” it is meant that the virus has the capacity to preferablytarget the cells of the liver either specifically or non-specifically.In further embodiments the virus is a modulated hepatitis virus, SV-40,or Epstein-Bar virus. In yet another embodiment, the virus is anadenovirus.

Gene Therapy

In one aspect of the invention a nucleic acid or nucleic acids encodinga PDX-1, Pax-4, MafA, NeuroD1, or Sox-9 polypeptide or a combinationthereof, or functional derivatives thereof, are administered by way ofgene therapy. Gene therapy refers to therapy that is performed by theadministration of a specific nucleic acid to a subject. In this aspectof the invention, the nucleic acid produces its encoded peptide(s),which then serve to exert a therapeutic effect by modulating function ofan aforementioned disease or disorder. e.g., diabetes. Any of themethodologies relating to gene therapy available within the art may beused in the practice of the present invention. See e.g., Goldspiel, etal., 1993. Clin Pharm 12: 488-505.

In a preferred embodiment, the therapeutic comprises a nucleic acid thatis part of an expression vector expressing any one or more of theaforementioned PDX-1, Pax-4, MafA, NeuroD1, and/or Sox-9 polypeptides,or fragments, derivatives or analogs thereof, within a suitable host. Ina specific embodiment, such a nucleic acid possesses a promoter that isoperably-linked to coding region(s) of a PDX-1, Pax-4, MafA, NeuroD1 andSox-9 polypeptide. The promoter may be inducible or constitutive, and,optionally, tissue-specific. The promoter may be, e.g., viral ormammalian in origin. In another specific embodiment, a nucleic acidmolecule is used in which coding sequences (and any other desiredsequences) are flanked by regions that promote homologous recombinationat a desired site within the genome, thus providing forintra-chromosomal expression of nucleic acids. See e.g., Koller andSmithies, 1989. Proc Natl Acad Sci USA 86: 8932-8935. In yet anotherembodiment the nucleic acid that is delivered remains episomal andinduces an endogenous and otherwise silent gene.

Delivery of the therapeutic nucleic acid into a patient may be eitherdirect (i.e., the patient is directly exposed to the nucleic acid ornucleic acid-containing vector) or indirect (i.e., cells are firstcontacted with the nucleic acid in vitro, then transplanted into thepatient). These two approaches are known, respectively, as in vivo or exvivo gene therapy. In a specific embodiment of the present invention, anucleic acid is directly administered in vivo, where it is expressed toproduce the encoded product. This may be accomplished by any of numerousmethods known in the art including, but not limited to, constructingsaid nucleic acid as part of an appropriate nucleic acid expressionvector and administering the same in a manner such that it becomesintracellular (e.g., by infection using a defective or attenuatedretroviral or other viral vector; see U.S. Pat. No. 4,980,286); directlyinjecting naked DNA; using microparticle bombardment (e.g., a “GeneGun®; Biolistic, DuPont); coating said nucleic acids with lipids; usingassociated cell-surface receptors/transfecting agents; encapsulating inliposomes, microparticles, or microcapsules; administering it in linkageto a peptide that is known to enter the nucleus; or by administering itin linkage to a ligand predisposed to receptor-mediated endocytosis(see, e.g., Wu and Wu, 1987. J Biol Chem 262: 4429-4432), which can beused to “target” cell types that specifically express the receptors ofinterest, etc.

An additional approach to gene therapy in the practice of the presentinvention involves transferring a gene into cells in in vitro tissueculture by such methods as electroporation, lipofection, calciumphosphate-mediated transfection, viral infection, or the like.Generally, the methodology of transfer includes the concomitant transferof a selectable marker to the cells. The cells are then placed underselection pressure (e.g., antibiotic resistance) so as to facilitate theisolation of those cells that have taken up, and are expressing, thetransferred gene. Those cells are then delivered to a patient. In aspecific embodiment, prior to the in vivo administration of theresulting recombinant cell, the nucleic acid is introduced into a cellby any method known within the art including, but not limited to:transfection, electroporation, microinjection, infection with a viral orbacteriophage vector containing the nucleic acid sequences of interest,cell fusion, chromosome-mediated gene transfer, microcell-mediated genetransfer, spheroplast fusion, and similar methodologies that ensure thatthe necessary developmental and physiological functions of the recipientcells are not disrupted by the transfer. See e.g., Loeffler and Behr,1993. Meth Enzymol 217: 599-618. The chosen technique should provide forthe stable transfer of the nucleic acid to the cell, such that thenucleic acid is expressible by the cell. Preferably, said transferrednucleic acid is heritable and expressible by the cell progeny. In analternative embodiment, the transferred nucleic acid remains episomaland induces the expression of the otherwise silent endogenous nucleicacid.

In preferred embodiments of the present invention, the resultingrecombinant cells may be delivered to a patient by various methods knownwithin the art including, but not limited to, injection of epithelialcells (e.g., subcutaneously), application of recombinant skin cells as askin graft onto the patient, and intravenous injection of recombinantblood cells (e.g., hematopoietic stem or progenitor cells) or livercells. The total amount of cells that are envisioned for use depend uponthe desired effect, patient state, and the like, and may be determinedby one skilled within the art.

Cells into which a nucleic acid can be introduced for purposes of genetherapy encompass any desired, available cell type, and may bexenogeneic, heterogeneic, syngeneic, or autogenic. Cell types include,but are not limited to, differentiated cells such as epithelial cells,endothelial cells, keratinocytes, fibroblasts, muscle cells, hepatocytesand blood cells, or various stem or progenitor cells, in particularembryonic heart muscle cells, liver stem cells (International PatentPublication WO 94/08598), neural stem cells (Stemple and Anderson, 1992,Cell 71: 973-985), hematopoietic stem or progenitor cells, e.g., asobtained from bone marrow, umbilical cord blood, peripheral blood, fetalliver, and the like. In a preferred embodiment, the cells utilized forgene therapy are autologous to the patient.

DNA for gene therapy can be administered to patients parenterally, e.g.,intravenously, subcutaneously, intramuscularly, and intraperitoneally.DNA or an inducing agent is administered in a pharmaceuticallyacceptable carrier, i.e., a biologically compatible vehicle which issuitable for administration to an animal e.g., physiological saline. Atherapeutically effective amount is an amount which is capable ofproducing a medically desirable result, e.g., an increase of apancreatic gene in a treated animal. Such an amount can be determined byone of ordinary skill in the art. As is well known in the medical arts,dosage for any given patient depends upon many factors, including thepatient's size, body surface area, age, the particular compound to beadministered, sex, time and route of administration, general health, andother drugs being administered concurrently. Dosages may vary, but apreferred dosage for intravenous administration of DNA is approximately10⁶ to 10²² copies of the DNA molecule. For example the DNA isadministers at approximately 2×10¹² virions per Kg.

Pharmaceutical Compositions

The compounds, e.g., PDX-1, Pax-4, MafA, NeuroD1, or Sox-9 polypeptides,nucleic acids encoding PDX-1, Pax-4, MafA, NeuroD1, or Sox-9polypeptides, or a nucleic acid or compound that increases expression ofa nucleic acid that encodes PDX-1, Pax-4, MafA, NeuroD1, or Sox-9polypeptides (also referred to herein as “active compounds”) of theinvention, and derivatives, fragments, analogs and homologs thereof, canbe incorporated into pharmaceutical compositions suitable foradministration. Such compositions typically comprise the nucleic acidmolecule, or protein, and a pharmaceutically acceptable carrier. As usedherein, “pharmaceutically acceptable carrier” is intended to include anyand all solvents, dispersion media, coatings, antibacterial andantifungal agents, isotonic and absorption delaying agents, and thelike, compatible with pharmaceutical administration. Suitable carriersare described in the most recent edition of Remington's PharmaceuticalSciences, a standard reference text in the field, which is incorporatedherein by reference. Preferred examples of such carriers or diluentsinclude, but are not limited to, water, saline, finger's solutions,dextrose solution, and 5% human serum albumin. Liposomes and non-aqueousvehicles such as fixed oils may also be used. The use of such media andagents for pharmaceutically active substances is well known in the art.Except insofar as any conventional media or agent is incompatible withthe active compound, use thereof in the compositions is contemplated.Supplementary active compounds can also be incorporated into thecompositions.

A pharmaceutical composition of the invention is formulated to becompatible with its intended route of administration. Examples of routesof administration include parenteral, e.g., intravenous, intradermal,subcutaneous, oral (e.g., inhalation), transdermal (topical),transmucosal, and rectal administration. Solutions or suspensions usedfor parenteral, intradermal, or subcutaneous application can include thefollowing components: a sterile diluent such as water for injection,saline solution, fixed oils, polyethylene glycols, glycerine, propyleneglycol or other synthetic solvents; antibacterial agents such as benzylalcohol or methyl parabens; antioxidants such as ascorbic acid or sodiumbisulfite; chelating agents such as ethylenediaminetetraacetic acid;buffers such as acetates, citrates or phosphates, and agents for theadjustment of tonicity such as sodium chloride or dextrose. The pH canbe adjusted with acids or bases, such as hydrochloric acid or sodiumhydroxide. The parenteral preparation can be enclosed in ampoules,disposable syringes or multiple dose vials made of glass or plastic.

Pharmaceutical compositions suitable for injectable use include sterileaqueous solutions (where water soluble) or dispersions and sterilepowders for the extemporaneous preparation of sterile injectablesolutions or dispersion. For intravenous administration, suitablecarriers include physiological saline, bacteriostatic water, CremophorEL™ (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In allcases, the composition must be sterile and should be fluid to the extentthat easy syringeability exists. It must be stable under the conditionsof manufacture and storage and must be preserved against thecontaminating action of microorganisms such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (for example, glycerol, propylene glycol, andliquid polyethylene glycol, and the like), and suitable mixturesthereof. The proper fluidity can be maintained, for example, by the useof a coating such as lecithin, by the maintenance of the requiredparticle size in the case of dispersion and by the use of surfactants.Prevention of the action of microorganisms can be achieved by variousantibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In manycases, it will be preferable to include isotonic agents, for example,sugars, polyalcohols such as mannitol, sorbitol, sodium chloride in thecomposition. Prolonged absorption of the injectable compositions can bebrought about by including in the composition an agent which delaysabsorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating the activecompound in the required amount in an appropriate solvent with one or acombination of ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the active compound into a sterile vehicle that contains abasic dispersion medium and the required other ingredients from thoseenumerated above. In the case of sterile powders for the preparation ofsterile injectable solutions, methods of preparation are vacuum dryingand freeze-drying that yields a powder of the active ingredient plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof.

Oral compositions generally include an inert diluent or an ediblecarrier. They can be enclosed in gelatin capsules or compressed intotablets. For the purpose of oral therapeutic administration, the activecompound can be incorporated with excipients and used in the form oftablets, troches, or capsules. Oral compositions can also be preparedusing a fluid carrier for use as a mouthwash, wherein the compound inthe fluid carrier is applied orally and swished and expectorated orswallowed. Pharmaceutically compatible binding agents, and/or adjuvantmaterials can be included as part of the composition. The tablets,pills, capsules, troches and the like can contain any of the followingingredients, or compounds of a similar nature: a binder such asmicrocrystalline cellulose, gum tragacanth or gelatin; an excipient suchas starch or lactose, a disintegrating agent such as alginic acid,Primogel, or corn starch; a lubricant such as magnesium stearate orSterotes; a glidant such as colloidal silicon dioxide; a sweeteningagent such as sucrose or saccharin; or a flavoring agent such aspeppermint, methyl salicylate, or orange flavoring.

Systemic administration can also be by transmucosal or transdermalmeans. For transmucosal or transdermal administration, penetrantsappropriate to the barrier to be permeated are used in the formulation.Such penetrants are generally known in the art, and include, forexample, for transmucosal administration, detergents, bile salts, andfusidic acid derivatives. Transmucosal administration can beaccomplished through the use of nasal sprays or suppositories. Fortransdermal administration, the active compounds are formulated intoointments, salves, gels, or creams as generally known in the art.

In one embodiment, the active compounds are prepared with carriers thatwill protect the compound against rapid elimination from the body, suchas a controlled release formulation, including implants andmicroencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid.Methods for preparation of such formulations will be apparent to thoseskilled in the art. The materials can also be obtained commercially fromAlza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions(including liposomes targeted to infected cells with monoclonalantibodies to viral antigens) can also be used as pharmaceuticallyacceptable carriers. These can be prepared according to methods known tothose skilled in the art, for example, as described in U.S. Pat. No.4,522,811, incorporated fully herein by reference.

It is especially advantageous to formulate oral or parenteralcompositions in dosage unit form for ease of administration anduniformity of dosage. Dosage unit form as used herein refers tophysically discrete units suited as unitary dosages for the subject tobe treated; each unit containing a predetermined quantity of activecompound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier. The specificationfor the dosage unit forms of the invention are dictated by and directlydependent on the unique characteristics of the active compound and theparticular therapeutic effect to be achieved.

The nucleic acid molecules of the invention can be inserted into vectorsand used as gene therapy vectors. Gene therapy vectors can be deliveredto a subject by any of a number of routes, e.g., as described in U.S.Pat. No. 5,703,055. Delivery can thus also include, e.g., intravenousinjection, local administration (see U.S. Pat. No. 5,328,470) orstereotactic injection (see e.g., Chen et al. (1994) PNAS 91:3054-3057).The pharmaceutical preparation of the gene therapy vector can includethe gene therapy vector in an acceptable diluent, or can comprise a slowrelease matrix in which the gene delivery vehicle is imbedded.Alternatively, where the complete gene delivery vector can be producedintact from recombinant cells, e.g., retroviral vectors, thepharmaceutical preparation can include one or more cells that producethe gene delivery system.

The pharmaceutical compositions can be included in a container, pack, ordispenser together with instructions for administration.

It should be understood that the present invention is not limited to theparticular methodologies, protocols and reagents, and examples describedherein. The terminology and examples used herein is for the purpose ofdescribing particular embodiments only, for the intent and purpose ofproviding guidance to the skilled arisan, and is not intended to limitthe scope of the present invention.

EXAMPLES Example 1 General Methods

Human Liver Cells

Adult human liver tissues were obtained from individuals 3-23 years oldor older. Liver tissues were used with the approval from the Committeeon Clinical Investigations (the institutional review board). Theisolation of human liver cells was performed as described (Sapir et al,2005; Meivar-Levy et al, 2007). The cells were cultured in Dulbecco'sminimal essential medium (1 g/l of glucose) supplemented with 10% fetalcalf serum, 100 units/ml penicillin; 100 ng/ml streptomycin; 250 ng/mlamphotericin B (Biological Industries, Beit Haemek, Israel), and kept at37° C. in a humidified atmosphere of 5% CO₂ and 95% air.

Viral Infection

The adenoviruses used in this study were as follows: Ad-CMV-Pdx-1 (Sapiret al, 2005; Meivar-Levy et al, 2007), Ad-RIP-luciferase (Seijffers etal, 1999), Ad-CMV-β-Gal, Ad-CMV-MafA (generous gift from Newgard, C. B.,Duke University), Ad-CMV-Pax4-IRES-GFP (generous gift from St Onge, L.DeveloGen AG, Gottingen, Germany), and Ad-CMV-Isl1 (generous gift fromKieffer, T. University of British Columbia, Vancouver, Canada). Theviral particles were generated by the standard protocol (He et al,1998).

Liver cells were infected with recombinant adenoviruses for 5-6 days(Table 1) supplemented with EGF (20 ng/ml; Cytolab, Ltd., Israel) andnicotinamide (10 mM; Sigma). The optimal multiplicity of infection (MOI)was determined according to cell survival (<75%) and induction ofc-peptide secretion. The MOI of the viruses used were; Ad-CMV-Pdx-1(1000 MOI), Ad-CMV-Pax4-IRES-GFP (100 MOI), Ad-CMV-MAf-A (10 MOI) andAd-CMV-Isl1 (100 MOI).

RNA Isolation, RT and RT-PCR Reactions

Total RNA was isolated and cDNA was prepared and amplified, as describedpreviously (Ber et al, 2003; Sapir et al, 2005). Quantitative real-timeRT-PCR was performed using ABI Step one plus sequence Detection system(Applied Biosystems, CA, USA), as described previously (Sapir et al,2005; Meivar-Levy et al, 2007; Aviv et al, 2009).

C-Peptide and Insulin Secretion Detection

C-peptide and insulin secretion were measured by static incubations ofprimary cultures of adult liver cells 6 days after the initial exposureto the viral treatment, as described (Sapir et al, 2005; Meivar-Levy etal, 2007; Aviv et al, 2009). The glucose-regulated c-peptide secretionwas measured at 2 mM and 17.5 mM glucose, which was determined bydose-dependent analyses to maximally induce insulin secretion fromtransdifferentiated liver cells, without having adverse effects ontreated cells function (Sapir et al, 2005; Meivar-Levy et al, 2007; Avivet al, 2009). C-peptide secretion was detected by radioimmunoassay usingthe human C-peptide radioimmunoassay kit (Linco Research, St. Charles,Mo.; <4% cross-reactivity to human proinsulin). Insulin secretion wasdetected by radioimmunoassay using human insulin radioimmunoassay kit(DPC, Angeles, Calif.; 32% cross-reactivity to human proinsulin). Thesecretion was normalized to the total cellular protein measured by aBio-Rad protein assay kit.

Luciferase Assay

Human liver cells were co-infected with Ad-RIP-luciferase (200moi) andthe various adenoviruses (as described below). Six days later,luciferase activity was measured using the Luciferase assay System(Promega) and the LKB 1250 Luminometer (LKB, Finland). The results werenormalized to total cellular protein measured by the Bio-Rad ProteinAssay kit (Bio-Rad).

Immunofluorescence

Human liver cells treated with the various adenoviruses, were plated onglass cover slides in 12-well culture plates (2×10⁵ cells/well). 3-4days later, the cells were fixed and stained as described (Sapir et al,2005; Meivar-Levy et al, 2007; Aviv et al, 2009). The antibodies used inthis study were: anti-rabbit PDX-1, anti-goat PDX-1 (both 1:1000 agenerous gift from C. V. E. Wright), anti-human insulin, anti-humansomatostatin (both 1:100, Dako, Glostrup, Denmark), anti-Pax4 (1:100;R&D Systems, Minneapolis, Minn.), anti-MafA (1:160; Santa CruzBiotechnology, Inc., Santa Cruz, Calif.). The secondary antibodies usedwere: anti-rabbit IgG Cyanine (cy2) conjugated antibody 1:250,anti-rabbit IgG indocarbocyanine (cy3) conjugated antibody 1:250,anti-goat IgG Cyanine (cy2) conjugated antibody 1:200, anti-goat IgGindocarbocyanine (cy3) conjugated antibody 1:250, and anti-mouse IgGindocarbocyanine (cy3) conjugated antibody 1:250 (all from JacksonImmunoResearch, PA). Finally, the cells were stained with4′,6-diamidino-2-phenyl-indole (DAPI, Sigma). The slides were imaged andanalyzed using a fluorescent microscope (Provis, Olympus).

Statistical Analysis

Statistical analyses were performed with a 2-sample Student t-testassuming unequal variances.

Example 2 Pdx-1-Induced Transdifferentiation

Previous studies (Sapir et al, 2005; Meivar-Levy et al, 2007; Aviv etal, 2009; Gefen-Halevi et al, 2010; Meivar-Levy et al, 2011) havesuggested that Pdx-1 alone is capable of inducing β-cell like phenotypeand function in human liver cells, possibly due to its capacity toactivate numerous otherwise silent endogenous pTFs in liver. Theactivation of the pancreatic lineage was fast and occurred within 5 days(Sapir et al, 2005, Ber et al, 2003)

In this example, the sequence of events that mediate Pdx-1 induced liverto pancreas transdifferentiation is examined Adenoviral vectors encodingPdx-1 were introduced to adult human liver cells, and the effects ofectopic Pdx-1 expression were monitored for four consecutive days postinfection (Days 2-5; FIG. 1). Pancreatic hormone and pancreas-specifictranscription factor expression was determined by quantitative RT-PCRanalysis every day for 5 days. Results were normalized to β-actin geneexpression within the same cDNA sample and are presented as the mean±SEof the relative expression versus control virus (Ad-CMV-β-gal, 1000 MOI)treated cells on the same day. Two independent experiments wereperformed, with n≧4, *p<0.05 and ** p<0.01.

Both glucagon and somatostatin genes were immediately activated, withinone day after Ad-Pdx-1 infection (FIGS. 1C and 1D). However, insulinexpression was only detected on the fourth to fifth day post-infection(FIG. 1A). To provide a mechanistic explanation for the distincttemporal activation of the three major pancreatic hormones, expressionlevels of endogenously activated transcription factors were analyzedduring the transdifferentiation process. The early pancreatic endocrinetranscription factors, NGN3 and NEUROD1 were immediately activated.However, β-cell specific TFs, such as NKX6.1 and MafA, were onlygradually and modestly activated in response to ectopic Pdx-1expression, reaching their peak expression level on the fourth and fifthday, respectively. The activation of insulin gene expression on thefifth day was associated not only with an increase in MafA expressionbut also with a decrease in Isl1 expression (FIG. 1D). These datasuggest that transdifferentiation of human liver cells along thepancreatic lineage, despite being rapid, is a gradual and consecutiveprocess. The distinct temporal activation of pancreatic hormone geneexpression (such as somatostatin and glucagon) can be partiallyattributed to the time course and the relative levels of theendogenously activated pTFs expression.

Example 3 Combined Expression of Pdx-1, Pax4 and Mafa Increases theEfficiency of Liver to Pancreas Transdifferentiation

Previous studies have suggested that the concerted expression of severalpTFs increases the transdifferentiation efficiency along the β-celllineage, compared to that induced by individual pTFs (Kaneto et al,2005; Tang et al, 2006; Song et al, 2007; Wang et al, 2007; Gefen-Haleviet al, 2010), as well as along other lineages. In order to analyze thisnotion in the experimental system of primary adult human liver cellcultures described herein, the individual and joint contribution ofthree major pTFs on liver to pancreas transdifferentiation wereinvestigated. Pdx-1, Pax4 and MafA, which mediate different stages inpancreas organogenesis, were ectopically co-expressed in primarycultures of adult human liver cells using recombinant adenoviruses.Cultured adult human liver cells were infected with Ad-CMV-Pdx-1 (1000MOI), Ad-CMV-Pax-4 (100 MOI) and Ad-CMV-MafA (10 MOI) alone or inconcert or with control virus (Ad-CMV-β-gal, 1000 MOI), and pancreaticdifferentiation markers were examined six days later. The multiplicityof infection (MOI) of each factor was titrated to result in maximalreprogramming efficiency associated by minimal adverse effects oninfected cell viability. Pdx-1 was expressed in 70% of the cells inculture, and the joint co-expression of all 3 pTFs was evident in 46.8%of the Pdx-1 positive cells (FIG. 2A). Very few cells stained positiveonly to Pax-4 or to MafA. Cells that stained positive for all three pTFsare indicated by the arrows (FIG. 2A, right panel). In FIG. 2B, livercells were co-infected with the combined pTFs and with Ad-RIP-LUC (200moi), and Luciferase activity of the insulin promoter was measured.

The combined expression of the three pTFs resulted in a substantialincrease in insulin promoter activation (FIG. 2B), a three-fold increasein the number of (pro)insulin producing cells (FIG. 2C) and 30-60%increase in glucose regulated (pro)insulin secretion (FIG. 2D), comparedto that induced by each of the pTFs alone. Taken together, these resultssuggest that the combination of the 3 pTFs increase transdifferentiationefficiency and also indicate that each of the factors is limited in itscapacity or is insufficient to individually promote maximaltransdifferentiation (Kaneto et al, 2005; Tang et al, 2006; Zhou et al,2008).

Example 4 Maturation and Segregation into the Different HormonesProducing Cells of Transdifferentiated Cells is Temporally Controlled inan Hierarchical Manner

In this example, the impact of temporally controlling the ectopic pTFsexpression was investigated to determine whether increasedtransdifferentiation efficiency by combined ectopic expression of thethree pTFs is also temporally controlled as suggested above (FIG. 2). Insupport of temporal control having a role in pancreastransdifferentiation, it is known that the three pTFs Pdx-1, Pax4, andMafA display distinct temporal expression and function during pancreasorganogenesis.

The three pTFs Pdx-1, Pax4, and MafA were introduced sequentially or inconcert to primary cultures of adult human liver cells using recombinantadenoviruses. Adenovirus-mediated ectopic gene expression peaks 17 hourspost infection (Varda-Bloom et al, 2001). Therefore, the pTFs weresequentially administered during three consecutive days (see Viralinfection in Example 1), allowing the manifestation of their individualeffects. Cells were infected according to the schedule as displayed inTable 1.

TABLE 1 Treatment order Day 1 Day 2 Day 3 Day 4 Day 5 Day 6 A Ad-β-galHarvest (control) B Ad-Pdx-1 + Harvest Ad-Pax4 + Ad-MafA C Ad-Pdx-1Ad-Pax4 Ad-Mafa Harvest D Ad-Mafa Ad-Pax4 Ad-Pdx-1 Harvest E Ad-Pdx1Ad-Mafa Ad-Pax4 Harvest

Cells were sequentially infected with one pTF adenoviral construct perday over three days in three different sequences: a direct hierarchicalorder (treatment C=Pdx-1→Pax4→MafA), in an opposite order (treatmentD=MafA→Pax4→Pdx-1), and in a random order (treatment E=Pdx-1→MafA→Pax4).The effect of the sequential pTFs administration on transdifferentiationefficiency and on the β-cell-like maturation was compared to that of theconcerted or simultaneous administration of all three pTFs on the firstday (treatment B=Pdx-1+Pax4+MafA) and to similar MOI of control virus(treatment A=β-gal) (Table 1 and FIG. 3A). Specifically, cultured adulthuman liver cells were infected with Ad-CMV-Pdx-1 (1000 MOI),Ad-CMV-Pax-4 (100 MOI) and Ad-CMV-MafA (10 MOI) together or in asequential manner as summarized in FIG. 3A and Table 1 (treatments B-E)or with control virus (Ad-CMV-β-gal, 1000 moi, treatment A), andanalyzed for their pancreatic differentiation six days later.

Insulin promoter activity (FIG. 4A), the percent of insulin producingcells (FIG. 3B) and glucose-regulated (pro)insulin secretion (FIG. 3C)were unaffected by the order of the sequentially administered pTFs.Interestingly, the sequential pTF administration in the random order(treatment E=Pdx-1→MafA→Pax4) resulted in increased insulin promoteractivity but was associated with loss of glucose regulation of insulinsecretion and decreased glucose transporter 2 (GLUT-2) expression (FIGS.3B, 3C and 4B). Loss of glucose sensing ability upon changing the orderof Pax4 and MafA administration suggests a potential effect of thesequence of expressed pTFs on β-cell-like maturation but not on theefficiency of the transdifferentiation process.

Example 5 Hierarchical Administration of Pdx-1, Pax4, and Mafa Promotesthe Maturation of Transdifferentiated Cells to β-Like Cells

The previous results encouraged further investigation to determine towhat extent and under which conditions increased transdifferentiationefficiency is associated with enhanced maturation along the β-celllineage. The hallmark characteristics of mature β-cells are the capacityto process the proinsulin and secrete it in a glucose-regulated manner(Eberhard et al, 2009; Borowiak, 2010). To analyze whether the temporalchanges in pTF expression distinctly affect transdifferentiated cellmaturation along the β-cell lineage, the effect of the distincttreatments A-E (Table 1 and FIG. 3A) on proinsulin processing andglucose-regulated c-peptide secretion was analyzed.

Indeed, only the direct hierarchical administration (treatment C) of thepTFs resulted in pronounced production of processed insulin and itsglucose-regulated secretion which displayed physiological glucose doseresponse characteristics (FIGS. 3C and 5A). The newly acquired phenotypeand function were stable, as demonstrated by the ability to secretec-peptide in a glucose-regulated manner for up to four weeks in vitro(FIGS. 5A and 5B).

The increased prohormone processing only upon the direct hierarchicalpTFs administration (treatment C) was associated with pronouncedincrease in PCSK2 and GLUT2 gene expression, which possess roles inprohormone processing and glucose sensing abilities, respectively (FIGS.3 and 4). These data suggest an obligatory role for the sequential anddirect hierarchical expression of pTFs in promoting the maturation andfunction of the transdifferentiated liver cells along the β-celllineage. Both concerted (treatment B) and sequential TF administrationin an indirect hierarchical mode (treatment D and E), failed to generatetransdifferentiated cells which display mature β-cell-likecharacteristics.

To provide a mechanistic explanation for the changes in the β-cell-likestate of maturation the repertoire of the endogenously activated pTFsunder the distinct temporal treatments (B-E) was analyzed. All thetreatments (B-E) resulted in increased expression of numerous endogenouspTFs (FIG. 3E), such as NEUROG3, NEUROD1, NKX6.1 and NKX2.2. However,the most robust difference between the “mature” (treatment C) and“immature” phenotypes (treatments B, E and D) was exhibited at thelevels of the endogenous Isl1 gene expression. Thus, the most enhancedmaturation along the β-cell lineage induced by direct hierarchical pTFsadministration (treatment C) correlates with a dramatic decrease inendogenous Isl1 expression (FIG. 3E, arrow). Taken together these datasuggest that the maturation of transdifferentiated cells to β cellscould be affected by the relative and temporal expression levels ofspecific pTFs.

Example 6 Hierarchical Administration of Pdx-1, Pax4, and Mafa Promotesthe Segregation of Transdifferentiated Cells Between β-Like and δ-LikeCells

Exclusion of MafA from treatment C (Table 1) induced both Isl-1 (FIG.6D) and somatostatin gene expression (FIG. 8D). To analyze whether Isl-1increased expression upon MafA exclusion indeed causes increasedSomatostatin gene expression, Ad-CMV-Isl-1 was added together with MafAon the 3^(rd) day (treatment C, in table 1). Indeed, Isl-1 increasedsomatostatin gene expression (FIG. 6E). Ectopic Isl-1 expression(C+Isl-1) caused also increased Somatostatin protein production (FIG.6F) and its co-production in insulin producing cells (FIG. 9 lowerpanel), suggesting that high MafA expression associated by low Isl-1expression is crucial for segregating between insulin and somatostatinproducing cells.

Example 7 Analysis of the Individual Contribution of Pdx-1, Pax4, andMafa to Liver to Pancreas Transdifferentiation

The sequential characteristics of the transdifferentiation process wereidentified by temporal gain of function studies. Further analysis of theseparate contribution of each of the transcription factors, Pdx-1, Pax4and MafA, to the hierarchical developmental process was performed by arelative and temporal “reduced function” approach. Adult human livercells were treated by the direct temporal and sequential reprogrammingprotocol (treatment C), from which one of the ectopic pTFs was omitted.The omitted pTF was replaced by a control adenovirus carrying β-galexpression at a similar multiplicity of infection. Specifically, adulthuman liver cells were treated by the direct “hierarchical” sequentialinfection order (treatment C, FIG. 3A and Table 1). One singletranscription factor (pTF) was omitted at a time and replaced byidentical moi of Ad-CMV-β-gal. Pdx-1 omission is indicated as (C-Pdx-1),Pax4 omission is indicated as (C-Pax4), and MafA omission is indicatedas (C-MafA).

The functional consequences of separately omitting each of the pTFs'expression were analyzed at the molecular and functional levels (FIG.6). Separate Pdx-1 and MafA omission (C-Pdx-1 and C-MafA, respectively)resulted in decreased insulin promoter activation (FIG. 6A), ablatedglucose response of processed insulin secretion (FIG. 6B) and decreasedGLUT2 and GK expression (FIG. 6C). Exclusion of MafA associated alsowith decreased expression of the prohormone convertase, PCSK2 (FIG. 6C).On the other hand, exclusion of Pax4 (C-Pax4) did not significantlyaffect insulin promoter activation, nor did it affect glucose-regulatedc-peptide secretion. Pax-4 omission was associated with decreased GLUT2and PCSK2 expression (FIG. 6C), possibly suggesting that the expressionof GK is sufficient for obtaining glucose control ability of the hormonesecretion.

Analysis of the consequences of the temporal and separate pTF exclusionon the repertoire of the endogenously activated pTF expression wasperformed to explain these developmental alterations. Pdx-1 and Pax4exclusion caused a marked decline in the expression of most other pTFs(including NeuroG3, NKX2.2, NKX6.2, and Pax6), suggesting that theirpotential contribution to increasing transdifferentiation efficiency isrelated to their capacity to activate endogenous pancreatic TFs (FIG.6D). On the other hand, exclusion of MafA did not contribute to furtheractivation of endogenous pTF expression, possibly reflecting its lateand restricted expression only in pancreatic β-cells. On the contrary,MafA contribution to increased insulin promoter activity, prohormoneprocessing and its glucose regulated secretion was associated only withdecreased Isl-1 expression (FIG. 6D). These data may suggest that MafAis not involved in further promoting the efficiency of endogenous pTFsexpression and liver to pancreas transdifferentiation, but rather inpromoting transdifferentiated cell maturation.

Example 8 Isl-1 Prevents Maturation of Transdifferentiated Cells to βCell Lineage

The effect of MafA on β-cell-like maturation may in part be associatedwith its capacity to repress Isl1 expression. To test this hypothesis,ectopic Isl1 was introduced by adenoviral infection (Ad-Isl1) intransdifferentiated cells. Briefly, adult human liver cells were treatedby the direct “hierarchical” sequential infection order (treatment C)and supplemented by Ad-Isl1 (1 or 100 MOI) at the 3^(rd) day (C+Is1).

As indicated above, the sequential administration of the three pTFs in adirect hierarchical manner (treatment C) resulted in both increasedtransdifferentiation efficiency and the maturation of the newlygenerated cells along the β-cell lineage. Isl1 was jointly administeredwith MafA on the third day (C+Is11). Indeed, Isl1 overexpression on thethird day, under the control of a heterologous promoter, resulted insubstantial decrease of insulin gene expression and ablation of glucoseregulated (pro)insulin secretion (FIG. 7). The loss of glucose-sensingability was associated with diminished GLUT2 expression (FIG. 7C). Theseresults suggest that deregulated Isl1 expression at the final stages ofthe transdifferentiation protocol potentially hampers the maturationalong the β cell lineage, and may account in part for the ablatedmaturation under low MafA expression.

Taken together, these data suggest a crucial obligatory role for directhierarchical expression of pTFs in promoting transdifferentiated livercell maturation along the β cell lineage. Moreover, the sequentialdevelopmental process is associated with both activation and repressionof pTFs that may promote or hamper transdifferentiated cell maturationalong the pancreatic β cell lineage.

Example 9 Pdx-1, Pax4 and Mafa Hierarchical Administration InducesGlucagon and Somatostatin Expression

Transdifferentiation along the endocrine pancreatic lineage results inthe activation of expression of numerous pancreatic hormones. The extentwith which these hormone expression levels are affected by the temporalmanipulation of the pTFs was also investigated. Gene expression ofpancreatic hormones glucagon (GCG) (FIGS. 8A and 8B), somatostatin (SST)(FIGS. 8A, 8D, and 8E) or a cells specific transcription factors (FIG.8C) were determined by quantitative real-time PCR analysis after theindicated treatments.

The transcription of both glucagon (GCG) and somatostatin (SST) geneswas induced by each of the individually expressed pTFs, mainly by Pdx-1and MafA and to a lower extent by Pax4 (FIG. 8A). A further increase inglucagon gene transcription occurred only upon the direct hierarchicaladministration of pTFs (FIG. 4A, see treatment C). Pdx-1 and MafAexerted their effects on glucagon expression in a process associatedwith the activation of the α-cell specific transcription factors ARX andBRAIN4 or ARX alone, respectively (FIG. 8C). Somatostatin geneexpression which remained unaffected by most treatments (FIGS. 8A and8D), was increased when the temporal protocol was concluded by ectopicPax4 expression (E=Pdx-1→MafA→Pax4). This sequential protocol alsoexhibited a deteriorative effect on glucose-regulated (pro)insulinsecretion and was associated by increased Isl1 endogenous expression(FIGS. 3C and E). The ablated maturation along the β cell lineage wasassociated with increased somatostatin gene expression and an increasednumber of somatostatin positive cells (FIG. 8F). Many of the cellsexhibited somatostatin and insulin co-localization (data not shown).

Exclusion of each pTF from the hierarchical administration (treatment C)as discussed in Example 6 was also utilized to further investigate therole of the individual pTFs in glucagon and somatostatin expression(FIGS. 8B and 8D). Pax4 exclusion substantially reduced somatostatingene expression, suggesting its potential role in inducing thetranscription of this gene (FIG. 8D). Interestingly, MafA exclusion atthe end of the developmental process also substantially increasedsomatostatin gene expression, suggesting a potential inhibitory effectof MafA on somatostatin gene expression. This effect could be alsoattributed to MafA's capacity to repress Isl1 expression. To addressthis hypothesis, the effect of ectopic Isl1 on somatostatin geneexpression was analyzed. Indeed, Ad-Isl1 administration on the third daytogether with MafA (C+Is11) increased somatostatin gene expression (FIG.8E), while decreasing insulin gene expression, hormone production andsecretion (FIGS. 8A, 8B and FIG. 7). Under these experimentalconditions, 40% of the insulin producing cells stained positive forsomatostatin with very few cells expressing somatostatin alone.

These results suggest that part of the maturation of trandifferentiatedcells to β-cells is attributed to MafA expression at the late stages ofthe transdifferentiation process. At this stage, MafA restrictssomatostatin expression in a process associated with its capacity toinhibit Isl1 expression.

FIG. 9 shows the proposed mechanism of pancreatic transcription factorinduced liver to pancreas transdifferentiation. Each of the pTFs iscapable of activating a modest β-cell-like phenotype, in a restrictednumber of human liver cells. The concerted expression of the pTFsmarkedly increases liver to endocrine pancreas transdifferentiation.However the newly generated cells are immature and coexpress bothinsulin and somatostatin. Only sequential administration of the samefactors in a direct hierarchical manner both increasestransdifferentiation efficiency and also the transdifferentiated cellmaturation along the β-cell lineage.

Example 10 Identification of Cell Populations with TransdifferentiationCapacity In Vivo

Cell populations with transdifferentiation capacity were identified invivo in mice. Ectopic expression of the Pdx-1 gene was achieved in micelivers. Despite the uniform expression of the ectopic Pdx-1 gene inabout 40-50% of the cells of the liver (FIG. 10A) (Ferber et al., NatMed. 2000, and Ber et al., JBC, 2003) insulin-producing cells (IPCs) inPdx-1-treated mice in vivo were primarily located close to central veins(FIG. 10B), which is characterized by active Wnt signaling and theexpression of glutamine synthetase (GS) (FIG. 1C). The co-localizationof GS expression and insulin activation by Pdx-1 also indicated thatthose cells that can activate the GSRE have a predisposition forincreased transdifferentiation capacity. Therefore, cell populationspredisposed for transdifferentiation can also be identified by GSREactivation and active Wnt-signaling pathway.

Example 10 Using Adenoviruses to Identify Human Liver Cells Predisposedfor Transdifferentiation

This example demonstrates the use of recombinant adenoviruses toidentify human liver cells that are predisposed fortransdifferentiation. Human liver cells in culture are heterogeneouswith regard to the activation of the intracellular Wnt signaling pathwayand expression of GS. As GS is uniquely expressed in pericentral livercells, therefore the capacity to activate GSRE (GS Regulatory Element)can be used as a selective parameter of isolation of relevant cells(Gebhardt et al., Prog Histochem Cytochem, 2007; Gebhardt et al.,Methods Mol Biol, 1998; and Gaunitz et al., Hepatology, 2005).

In addition as the GSRE contains also a STAT3 binding element, thepredisposition of the cells to transdifferentiation could be mediated bythis element. The STAT3 pathway could also be involved in endowing thecells with reprogramming or transdifferentiation predisposition (FIGS.10, 11, 14 and 19).

Example 11 GSRE Repetitively Targets 13-15% of the Human Liver Cells inCulture

GSRE includes TCF/LEF and STATS binding elements (FIG. 11). Tworecombinant adenoviruses which carry the expression of eGFP gene orPdx-1 genes under the control of GSRE (FIG. 11) operatively linked to aminimal TK promoter (FIG. 11) have been generated. These adenovirusesdrove the expression of either Pdx-1 (FIG. 12A) or eGFP (FIG. 12B). Bothproteins were repetitively expressed in about 13-15% of the human livercells in culture suggesting the targeting of a specific population ofliver cells.

Example 12 GSRE Driven PDX-1 is More Efficient than CMV Driven PDX-1 inActivating Insulin Production in Liver Cells

Despite the repetitive expression of GSRE driven PDX-1 only in about13±2% of the cells in cultureits transdifferentiation capacity wassimilar or higher than that induced by Ad-CMV-Pdx-1, which drives Pdx-1expression in 60-80% of the cells in culture (FIG. 13). GSRE-activatingcells could account for most of the transdifferentiation capacity of theentire adult human liver cells in culture. Insulin production occurredin 25% of Pdx-1 positive cells upon Ad-GSRE-Pdx-1 treatment compared to1% of the Ad-CMV-Pdx-1 treated cells.

Example 13 Using Lentiviruses to Permanently Label the GSRE+ Cells byEGFP

Permanent lineage tracing was performed using Lentivirus constructs. Invitro lineage tracing for GSRE activity was performed by a modified duallentivirus system recently used to trace KRTS in keratinocytes(Mauda-Havakuk, et al., PLoS One, 2011) or albumin (Meivar-Levy et al.,J Transplant, 2011) expression in liver cells. This lentivirus system (acollaboration with Prof. P. Ravassard from Université Pierre et MarieCurie Paris, France; FIG. 12A) includes the CMV-loxP-DsRed2-loxP-eGFP(R/G) reporter (Meivar-Levy et al., J Transplan, 2011; Mauda-Havakuk etal., PLoS One, 2011; and Russ et al., Diabetes, 2008) and an additionallentiviral vector carrying the expression of Cre recombinase under thecontrol of GSRE and a minimal TK promoter (generously contributed byProf. Gaunitz, (Gebhardt et al., Prog Histochem Cytochem, 2007 andGaunitz et al., Hepatology, 2005) Germany, FIG. 3A). Thus,GSRE-activating cells are irreversibly marked by eGFP (eGFP+), while therest of the doubly infected cells are marked by DsRed2 (DsRed2+). Ten tofourteen percent of the cells became eGFP+ within less than 10 days(FIG. 14B). The cells were separated by a cell sorter (FIG. 14) andseparately propagated (FIG. 15A). Cultures of eGFP+ (GSRE activators)and DsRed2+ cells were generated from 10 different human donors (ages3-60).

Example 14 EGFP+ Cells Consistently Exhibited SuperiorTransdifferentiation Capacity

Human liver cells separated by lineage tracing according to GSREactivity efficiently propagated (FIG. 15A) and were similarlyefficiently infected by recombinant adenoviruses. eGFP+ cellsconsistently exhibited superior transdifferentiation capacity (FIG. 16)manifested by insulin and glucagon gene expression which was comparableto that of human pancreatic islets in culture (FIG. 16A), glucoseregulated insulin secretion (FIG. 16B) and glucose regulated C-peptidesecretion (FIG. 16C). These capacities were consistant and did notdiminished upon extensive cell proliferation, (FIG. 17).

Example 15 Characterization of Cells with Predisposition forTransdifferentiation

To identify the factors which could potentially affect the distincttransdifferentiation efficiencies of the human liver cells, we comparedthe global gene expression profile of the two separated populationsusing microarray chip analyses. Human liver cell cultures derived from 3different donors and separated into eGFP+ and DsRed2+ cells andpropagated for 4 passages. The extracted RNA was converted into cDNA andsubjected to microarray chip analysis using the General Human Array(GeneChip Human Genome U133A 2.0 Array, Affymetrix). While most of thegenes were expressed at comparable levels in the separated groups, theexpression of about 800 probes was significantly different (FIG. 18).According to microarray chip analyses, about 100 genes coding formembrane proteins are differentially expressed between thetransdifferentiation-prone (eGFP+) and non-responding (DsRed2+) cells.Several of these markers are presented in Table 2.

TABLE 2 Membrane antigens that are differentially expressed in eGFP+ andDsRed2+ cells. High Fold Antigene expression (Log 2) p-value commercialantibody ABCB1 DsRed2 −6.363 1.52E−02 BD Biosciences (#557002) ITGA4DsRed2 −1.979 2.69E−02 R&D system (FAB1354G) ABCB4 DsRed2 −4.42 4.62E−02Abcam (ab24108) PRNP DsRed2 −1.35 4.20E−02 eBioscience (12-9230-73)HOMER1 eGFP 1.41 3.25E−04 Biorbyt(orb37754) LAMP3 eGFP 1.285 1.81E−02 BDBiosciences (#558126) BMPR2 eGFP 1.236 3.50E−02 R&D system (AF811)

Microarray data suggested numerous membrane proteins that aredifferential expression between the eGFP+ and the DsRed2+ cells(Fold=eGFP+ differential expression compared to the DsRed2+ (log 2)).All the presented antigens have commercially available antibodies.

Example 16 Wnt Signaling is Active in Cells Predisposed forTransdifferentiation

Liver zonation has been suggested to be controlled by a gradient ofactivated β-catenin levels; while most cells in the liver contain verylow β-catenin activity, the pericentral liver cells express highβ-catenin activity associated with active Wnt signaling (Gebhardt, etal., Prog Histochem Cytochem, 2007). Since Wnt signaling is obligatoryfor competent 13 cell activity (Liu et al., J Biol Chem, 2008; Liue etal., Adv Exp Med Biol, 2010; Loder et al., Biochem Soc Trans, 2008; andShu et al., Diabetes, 2008), the pTFs-induced pancreatic lineageactivation in the liver is restricted to cells that a priori displayactive Wnt signaling.

GSRE utilized a TCF regulatory element isolated from the 5′ enhancer ofGS. If Pdx-1-induced liver to pancreas transdifferentiation is mediatedin part by the intracellular Wnt signaling pathway, factors whichmodulate the Wnt signaling pathway should also affecttransdifferentiation efficiency (FIG. 19).

This data in adult human liver cells suggest that increasingconcentrations of Wnt3a increased Pdx-1-induced glucose-regulatedinsulin secretion, while DKK3 (an inhibitor of the Wnt signalingpathway) completely abolished the effect of Pdx-1 on the process (FIG.19). DKK3 also totally abolished the transdifferentiation capacity ofthe eGFP cells isolated according to their ability to activate GSRE(FIG. 20).

Characterization of Wnt signaling pathway activity in the eGFP+ andDsRed+ cell populations was performed. The APC expression, whichparticipates in 3-catenin destabilization, thus diminishing Wntsignaling, was 700% higher in DsRed2+ cells than in the eGFP+ cells(FIG. 21A, in relative agreement with the zonation displayed in vivo).The eGFP+ population has increased activated β-catenin levels (40%)compared to the levels analyzed in DsRed2+ cells (FIGS. 21B and C).These data demonstrate that Wnt signaling is active in cells that arecompetent for GSRE activation and have predisposition fortransdifferentiation.

Other Embodiments

While the invention has been described in conjunction with the detaileddescription thereof, the foregoing description is intended to illustrateand not limit the scope of the invention, which is defined by the scopeof the appended claims. Other aspects, advantages, and modifications arewithin the scope of the following claims.

REFERENCES

-   1. Ambasudhan, R., M. Talantova, et al. (2011). Direct reprogramming    of adult human fibroblasts to functional neurons under defined    conditions. Cell 9: 113-118.-   2. Atala, A. (2008). Extending life using tissue and organ    replacement. Curr Aging Sci 1: 73-83.-   3. Aviv, V., I. Meivar-Levy, et al. (2009). Exendin-4 promotes liver    cell proliferation and enhances PDX-1-induced liver to pancreas    transdifferentiation. J Biol Chem 284: 33509-33520.-   4. Ber, I., K. Shternhall, et al. (2003). Functional, persistent,    and extended liver to pancreas transdifferentiation. J Biol Chem    278: 31950-31957.-   5. Bernardo, A. S., C. W. Hay, et al. (2008). Pancreatic    transcription factors and their role in the birth, life and survival    of the pancreatic beta cell. Mol Cell Endocrinol 294: 1-9.-   6. Bonal, C. and P. L. Herrera (2008). Genes controlling pancreas    ontogeny. Int J Dev Biol 52: 823-835.-   7. Borowiak, M. (2010). The new generation of beta-cells:    replication, stem cell differentiation, and the role of small    molecules. Rev Diabet Stud 7: 93-104.-   8. Brun, T. and B. R. Gauthier (2008). A focus on the role of Pax4    in mature pancreatic islet beta-cell expansion and survival in    health and disease. J Mol Endocrinol 40: 37-45.-   9. Chakrabarti, S. K. and R. G. Mirmira (2003). Transcription    factors direct the development and function of pancreatic b-cells.    Trends Endocrinol Metab 14: 78-84.-   10. Collombat, P., J. Hecksher-Sorensen, et al. (2006). Specifying    pancreatic endocrine cell fates. Mech Dev 123: 501-512.-   11. Collombat, P., A. Mansouri, et al. (2003). Opposing actions of    Arx and Pax4 in endocrine pancreas development. Genes Dev 17:    2591-2603.-   12. D'Amour, K. A., A. D. Agulnick, et al. (2005). Efficient    differentiation of human embryonic stem cells to definitive    endoderm. Nat Biotechnol. 23: 1534-1541.-   13. Eberhard, D. and E. Lammert (2009). The pancreatic beta-cell in    the islet and organ community. Curr Opin Genet Dev 19: 469-475.-   14. Ferber, S., A. Halkin, et al. (2000). Pancreatic and duodenal    homeobox gene 1 induces expression of insulin genes in liver and    ameliorates streptozotocin-induced hyperglycemia. Nat Med 6:    568-572.-   15. Gefen-Halevi, S., I. H. Rachmut, et al. (2010). NKX6.1 promotes    PDX-1-induced liver to pancreatic beta-cells reprogramming Cell    Reprogram 12: 655-664.-   16. Gradwohl, G., A. Dierich, et al. (2000). neurogenin3 is required    for the development of the four endocrine cell lineages of the    pancreas. Proc Natl Acad Sci USA 97: 1607-1611.-   17. Hanna, J., S. Markoulaki, et al. (2008). Direct reprogramming of    terminally differentiated mature B lymphocytes to pluripotency. Cell    133: 250-264.-   18. He, T. C., S. Zhou, et al. (1998). A simplified system for    generating recombinant adenoviruses. Proc Natl Acad Sci USA 95:    2509-2514.-   19. Ieda, M., J. D. Fu, et al. (2010). Direct reprogramming of    fibroblasts into functional cardiomyocytes by defined factors. Cell.    142: 375-386.-   20. Iwasaki, H., S. Mizuno, et al. (2006). The order of expression    of transcription factors directs hierarchical specification of    hematopoietic lineages. Genes Dev 20: 3010-3021.-   21. Kaneto, H., T. A. Matsuoka, et al. (2005). A crucial role of    MafA as a novel therapeutic target for diabetes. J Biol Chem 280:    15047-15052.-   22. Kaneto, H., Y. Nakatani, et al. (2005). PDX-1/VP16 fusion    protein, together with NeuroD or Ngn3, markedly induces insulin gene    transcription and ameliorates glucose tolerance. Diabetes 54:    1009-1022.-   23. Kataoka, K., S. I. Han, et al. (2002). MafA is a    glucose-regulated and pancreatic beta-cell-specific transcriptional    activator for the insulin gene. J Biol Chem 277: 49903-49910.-   24. Koizumi, M., R. Doi, et al. (2004). Hepatic regeneration and    enforced PDX-1 expression accelerate transdifferentiation in liver.    Surgery 136: 449-457.-   25. Kojima, H., M. Fujimiya, et al. (2003). NeuroD-betacellulin gene    therapy induces islet neogenesis in the liver and reverses diabetes    in mice. Nat Med 9: 596-603.-   26. Kroon, E., L. A. Martinson, et al. (2008). Pancreatic endoderm    derived from human embryonic stem cells generates glucose-responsive    insulin-secreting cells in vivo. Nat Biotechnol 26: 443-452.-   27. Meivar-Levy, I. and S. Ferber (2003). New organs from our own    tissues: liver-to-pancreas transdifferentiation. Trends Endocrinol    Metab 14: 460-466.-   28. Meivar-Levy, I. and S. Ferber (2006). Regenerative medicine:    using liver to generate pancreas for treating diabetes. Isr Med    Assoc J. 8: 430-434.-   29. Meivar-Levy, I. and S. Ferber (2010). Adult cell fate    reprogramming converting liver to pancreas. Methods Mol. Biol. 636:    251-283.-   30. Meivar-Levy, I., T. Sapir, et al. (2011). Human liver cells    expressing albumin and mesenchymal characteristics give rise to    insulin-producing cells. J Transplant 2011:252387.

31. Meivar-Levy, I., T. Sapir, et al. (2007). Pancreatic and duodenalhomeobox gene 1 induces hepatic dedifferentiation by suppressing theexpression of CCAAT/enhancer-binding protein beta. Hepatology 46:898-905.

-   32. Murtaugh, L. C. and D. A. Melton (2003). Genes, signals, and    lineages in pancreas development. Annu Rev Cell Dev Biol 19: 71-89.-   33. Nishimura, W., S. Bonner-Weir, et al. (2009). Expression of MafA    in pancreatic progenitors is detrimental for pancreatic development.    Dev Biol 333: 108-120.-   34. Offield, M. F., T. L. Jetton, et al. (1996). PDX-1 is required    for pancreatic outgrowth and differentiation of the rostral    duodenum. Development 122: 983-995.-   35. Olbrot, M., J. Rud, et al. (2002). Identification of    beta-cell-specific insulin gene transcription factor RIPE3b1 as    mammalian MafA. Proc Natl Acad Sci USA 99: 6737-6742.-   36. Pang, Z. P., N. Yang, et al. (2011). Induction of human neuronal    cells by defined transcription factors. Nature 476: 220-223.-   37. Russ, H. A. and S. Efrat (2011). Development of human    insulin-producing cells for cell therapy of diabetes. Pediatr    Endocrinol Rev 9: 590-597.-   38. Sapir, T., K. Shternhall, et al. (2005). From the Cover:    Cell-replacement therapy for diabetes: Generating functional    insulin-producing tissue from adult human liver cells. Proc Natl    Acad Sci USA 102: 7964-7969.-   39. Seijffers, R., O. Ben-David, et al. (1999). Increase in PDX-1    levels suppresses insulin gene expression in RIN 1046-38 cells.    Endocrinology 140: 3311-3317.-   40. Sheyn, D., O. Mizrahi, et al. (2010). Genetically modified cells    in regenerative medicine and tissue engineering. Adv Drug Deliv Rev    62: 683-698.-   41. Slack, J. M. and D. Tosh (2001). Transdifferentiation and    metaplasia—switching cell types. Curr Opin Genet Dev 11: 581-586.-   42. Song, Y. D., E. J. Lee, et al. (2007). Islet cell    differentiation in liver by combinatorial expression of    transcription factors neurogenin-3, BETA2, and RIPE3b1. Biochem    Biophys Res Commun. 354: 334-339.-   43. Stoffers, D. A., M. K. Thomas, et al. (1997). The homeodomain    protein IDX-1. Trends Endocrinol. & Metab. 8: 145-151.-   44. Szabo, E., S. Rampalli, et al. (2010). Direct conversion of    human fibroblasts to multilineage blood progenitors. Nature 468:    521-526.-   45. Takahashi, K. and S. Yamanaka (2006). Induction of pluripotent    stem cells from mouse embryonic and adult fibroblast cultures by    defined factors. Cell 126: 663-676.-   46. Tang, D. Q., L. Z. Cao, et al. (2006). Role of Pax4 in    Pdx1-VP16-mediated liver-to-endocrine pancreas transdifferentiation.    Lab Invest. 86: 829-841.-   47. Varda-Bloom, N., A. Shaish, et al. (2001). Tissue-specific gene    therapy directed to tumor angiogenesis. Gene Ther 8: 819-827.-   48. Vierbuchen, T., A. Ostermeier, et al. (2010). Direct conversion    of fibroblasts to functional neurons by defined factors. Nature 463:    1035-1041.-   49. Wang, A. Y., A. Ehrhardt, et al. (2007). Adenovirus Transduction    is Required for the Correction of Diabetes Using Pdx-1 or    Neurogenin-3 in the Liver. Mol Ther 15: 255-263.-   50. Yamanaka, S. (2008). Induction of pluripotent stem cells from    mouse fibroblasts by four transcription factors. Cell Prolif. 41:    51-56.-   51. Yechoor, V. and L. Chan (2010). Minireview: beta-cell    replacement therapy for diabetes in the 21st century: manipulation    of cell fate by directed differentiation. Mol Endocrinol 24:    1501-1511.-   52. Zhou, Q., J. Brown, et al. (2008). In vivo reprogramming of    adult pancreatic exocrine cells to beta-cells. Nature 455: 627-632.

1.-63. (canceled)
 64. A method of producing a transdifferentiatedpopulation of cells having a mature pancreatic beta cell phenotype andfunction comprising: (a) contacting an adult mammalian non-pancreaticbeta cell population with a pancreatic and duodenal homeobox (PDX-1)polypeptide or a nucleic acid encoding a pancreatic and duodenalhomeobox (PDX-1) polypeptide under conditions to allow uptake of thesaid polypeptides or nucleic acids at a first time period; (b)contacting the population of cells of step (a) with a Pax-4 polypeptide,or a NeuroD1 polypeptide, or a nucleic acid encoding a Pax-4polypeptide, or a nucleic acid encoding a NeuroD1 polypeptide underconditions to allow uptake of the said polypeptides or nucleic acids ata second time period; and (c) contacting the population of cells of step(b) with a MafA polypeptide or a nucleic acid encoding a MafApolypeptide under conditions to allow uptake of the polypeptide or thenucleic acid at a third time period; thereby producing atransdifferentiated population of cells having a mature pancreatic betacell phenotype and function.
 65. The method of claim 64, wherein saidnon-pancreatic beta cell population is enriched for cells predisposed totransdifferentiation.
 66. The method of claim 65, wherein saidpredisposed cells acquire a pancreatic beta cell phenotype and functionupon ectopic administration of pancreatic transcription factors.
 67. Themethod of claim 64, wherein the second time period is at least 24 hoursafter the first time period or is at the same time as the first timeperiod, and wherein the third time period is at least 24 hours after thesecond time period.
 68. The method of claim 64, wherein the populationof adult mammalian cells is selected from the group consisting of: bonemarrow, muscle, spleen, kidney, blood, skin, pancreas, and liver cells.69. The method of claim 68, wherein said liver cells comprise anenriched population of liver cells predisposed to transdifferentiation.70. The method of claim 69, wherein said predisposed liver cellscomprise pericentral liver cells.
 71. The method of claim 64, whereinthe population of cells is contacted in vivo.
 72. The method of claim64, wherein the population of cells is contacted in vitro.
 73. Themethod of claim 65, wherein said population of cells predisposed totransdifferentiation comprises cells comprising: (a) an activeWnt-signaling pathway; (b) a capability of activating the glutaminesynthetase response element (GSRE); (c) increased expression of HOMER1,LAMP3 or BMPR2, or any combination thereof; (d) decreased expression ofABCB1, ITGA4, ABCB4, or PRNP, or any combination thereof; or anycombination thereof.
 74. A population of transdifferentiated cellshaving a mature pancreatic beta cell phenotype and function producedfrom an adult mammalian non-pancreatic beta cell population.
 75. Thepopulation of transdifferentiated cells of claim 74, wherein saidnon-pancreatic beta cell population is predisposed totransdifferentiation.
 76. The population of transdifferentiated cells ofclaim 74, wherein said mature pancreatic beta cell phenotype andfunction comprises increased glucose regulated processed insulinsecretion.
 77. The population of transdifferentiated cells of claim 74,produced by a method comprising: (a) contacting the adult mammaliannon-pancreatic beta cell population with a pancreatic and duodenalhomeobox (PDX-1) polypeptide or a nucleic acid encoding a pancreatic andduodenal homeobox (PDX-1) polypeptide under conditions to allow uptakeof the said polypeptides or nucleic acids at a first time period; (b)contacting the population of cells of step (a) with a Pax-4 polypeptide,or a NeuroD1 polypeptide, or a nucleic acid encoding a Pax-4polypeptide, or a nucleic acid encoding a NeuroD1 polypeptide underconditions to allow uptake of the said polypeptides or nucleic acids ata second time period; and (c) contacting the population of cells of step(b) with a MafA polypeptide or a nucleic acid encoding a MafApolypeptide under conditions to allow uptake of the polypeptide or thenucleic acid at a third time period.
 78. The population oftransdifferentiated cells of claim 77, wherein the second time period isat least 24 hours after the first time period or is at the same time asthe first time period, and wherein the third time period is at least 24hours after the second time period.
 79. The population oftransdifferentiated cells of claim 74, wherein said population of adultmammalian non-pancreatic cells is selected from the group consisting of:bone marrow cells, muscle cells, spleen cells, kidney cells, bloodcells, skin cells, and liver cells.
 80. The population oftransdifferentiated cells of claim 79, wherein the population of livercells comprises an enriched population of pericentral liver cells. 81.The population of transdifferentiated cells of claim 75, wherein saidpopulation of predisposed cells comprises cells comprising: (a) anactive Wnt-signaling pathway; (b) a capability of activating theglutamine synthetase response element (GSRE); (c) increased expressionof HOMER1, LAMP3 or BMPR2, or any combination thereof; (d) decreasedexpression of ABCB1, ITGA4, ABCB4, or PRNP, or any combination thereof;or any combination thereof.
 82. A method of treating a degenerativepancreatic disorder in a subject in need thereof, said method comprisingadministering to said subject: (a) a composition comprising a PDX-1polypeptide or a nucleic acid encoding a PDX-1 polypeptide at a firsttime period, (b) a composition comprising a Pax-4 polypeptide, or aNeuroD1 polypeptide, or a nucleic acid encoding a Pax-4 polypeptide or anucleic acid encoding a NeuroD1 polypeptide at a second time period; and(c) a composition comprising a MafA polypeptide or a nucleic acidencoding a MafA polypeptide at a third time period; thereby treatingsaid degenerative pancreatic disorder.
 83. The method of claim 82,wherein the second time period is at least 24 hours after the first timeperiod or is at the same time as the first time period, and wherein thethird time period is at least 24 hours after the second time period. 84.The method of claim 82, wherein the degenerative pancreatic disorder isdiabetes.
 85. The method of claim 84, wherein the diabetes is Type I orType II diabetes.
 86. The method of claim 82, wherein the degenerativepancreatic disorder is pancreatic cancer or pancreatitis.
 87. A methodof treating a degenerative pancreatic disorder in a subject in needthereof, said method comprising administering to said subject atransdifferentiated population of adult mammalian non-pancreatic betacells, said transdifferentiated population comprising a maturepancreatic beta cell phenotype and function, thereby treating saiddegenerative pancreatic disorder.
 88. The method of claim 87, whereinthe degenerative pancreatic disorder is diabetes.
 89. The method ofclaim 88, wherein the diabetes is Type I or Type II diabetes.
 90. Themethod of claim 87, wherein the degenerative pancreatic disorder ispancreatic cancer or pancreatitis.
 91. A method of isolating apopulation of cells that have an enriched capacity for transcriptionfactor induced transdifferentiation, said method comprising the stepsof: (a) providing a heterogeneous population of human cells; (b)introducing into said cells a nucleic acid construct comprising aglutamine synthetase response element (GSRE) or fragment thereof capableof activating glutamine synthetase transcription, operatively linked toa reporter protein; (c) isolating the cells expressing the reporterprotein; thereby isolating a cell population with enrichedtransdifferentiation capacity.
 92. The method of claim 91, wherein inthe nucleic acid construct further comprises a promoter/enhancer. 93.The method of claim 91, wherein the reporter protein provides resistanceto selection pressure.
 94. The method of claim 91, wherein the cells areendothelial cells, fibroblasts, mesenchymal or liver cells.
 95. Themethod of claim 94, wherein the liver cells are pericentral liver cells.96. An isolated population of cells having enriched transdifferentiationcapacity, isolated by the method of: (a) providing a heterogeneouspopulation of human cells; (b) introducing into said cells a nucleicacid construct comprising a glutamine synthetase response element (GSRE)or fragment thereof capable of activating glutamine synthetasetranscription, operatively linked to a reporter protein; and (c)isolating the cells expressing the reporter protein; wherein theisolated cell population comprises cells comprising an enrichedtransdifferentiation capacity.
 97. A method of increasingtransdifferentiation efficiency in a population of cells comprisingtransdifferentiating the isolated cell population of claim
 96. 98. Apopulation of liver cells enriched for cells predisposed totransdifferentiation, said cells comprising: (a) an active Wnt-signalingpathway; (b) a capability of activating the glutamine synthetaseresponse element (GSRE); (c) increased expression of HOMER1, LAMP3 orBMPR2, or any combination thereof; (d) decreased expression of ABCB1,ITGA4, ABCB4, or PRNP, or any combination thereof; or any combinationthereof.
 99. The population of liver cells of claim 98, wherein ectopicexpression of at least one pancreatic transcription factor in saidcells, activates the pancreatic beta cell lineage, and activates insulinproduction and secretion from said cells.
 100. The population of livercells of claim 98, wherein said cells comprise at least an activeWnt-signaling pathway and at least one other element selected from (b),(c) and (d).